Dyes and labeled molecules

ABSTRACT

Dimeric and trimeric nucleic acid dyes, and associated systems and methods are provided. Such a dye may form a hairpin-like structure that enables it to stain nucleic acids via a release-on-demand mechanism, for example. Such a dye may have low background fluorescence in the absence of nucleic acids and high fluorescence in the presence of nucleic acids, upon binding therewith, for example. A dye provided herein may be useful in a variety of applications, such as in DNA quantitation in real-time PCR, for example.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation application and claims priority toU.S. application Ser. No. 11/377,253, filed Mar. 16, 2006, which claimsthe benefit of U.S. Provisional Application No. 60/663,613, filed onMar. 17, 2005, the entire contents of which are incorporated herein intheir entirety by this reference.

STATEMENT REGARDING NAMES OF PARTIES TO A JOINT RESEARCH AGREEMENT

Biotium, Inc. (Hayward, Calif. (CA)) and AlleLogic Biosciences Corp.(Hayward, Calif.) are parties to a joint research agreement pertainingto the present invention.

REFERENCE TO A SEQUENCE LISTING AND REQUEST AND INCORPORATION BYREFERENCE CONCERNING SAME

An original ASCII diskette and another ASCII diskette, which was aduplicate of the original diskette, containing the Sequence Listing forSEQ ID NO: 1 through SEQ ID NO: 10 disclosed herein, as well as a papercopy of the Sequence Listing, were submitted in, referred to in, andincorporated in their entireties, including the contents thereof, byreference in, the aforementioned U.S. Provisional Application No.60/663,613. A paper copy of the Sequence Listing is submitted herewith.It is hereby requested that the compliant computer readable SequenceListing that is already on file for the aforementioned U.S. ProvisionalApplication No. 60/663,613 be used in connection with this application.The paper or compact disc copy of the Sequence Listing in thisapplication, and the content thereof, are identical to the computerreadable copy of the Sequence Listing that was filed for theaforementioned U.S. Provisional Application No. 60/663,613. The SequenceListing, the paper copy of the Sequence Listing that is submittedherewith, and the computer readable Sequence Listing that is already onfile for the aforementioned U.S. Provisional Application No. 60/663,613are hereby incorporated herein, in their entireties, including thecontents thereof, by this reference.

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety.

BACKGROUND

Fluorescent dyes have been used for the detection and analysis ofbiological samples. As fluorescent dyes are highly sensitive, they canbe used to detect a very small number of fluorescent molecules. Forexample, such fluorescent dyes can be used to detect fewer than 50fluorescent molecules that are associated with cells. Barak, et al., J.Cell Biol. 90, 595 (1981).

Fluorescent dyes may be used as probes for use in imaging in live cellsor tissue samples. For example, a fluorescent-dye probe bound to areceptor on the surface of Dictyostelium cells has been used in theimaging of a single molecule of fluorescently labeled cAMP. Ueda, etal., Science 294, 864 (2001). Several fluorescent probes havingdifferent fluorescent wavelengths may be used to perform multi-colorimaging in live cells or tissue samples. Fluorescent probes are highlysensitive, of relatively low toxicity, and easy to dispose of relativeto radioactive probes.

Fluorescent dyes can be used in the detection of nucleic acids,including DNA and RNA, and biological samples involving nucleic acids.Nucleic acid polymers such as DNA and RNA are involved in thetransmission of genetic information from one generation to the next andto the routine functioning of living organisms. Nucleic acids are thusof interest and the objects of study. Fluorescent nucleic acid dyes thatspecifically bind to nucleic acids and form highly fluorescent complexesare useful tools for such study. These dyes can be used to detect thepresence and quantities of DNA and RNA in a variety of media, includingpure solutions, cell extracts, electrophoretic gels, micro-array chips,live or fixed cells, dead cells, and environmental samples. These dyescan be used in the quantitative detection of DNA in real-time polymerasechain reaction (qPCR), which is a technique used in genomic research andmedical diagnosis.

Polymerase chain reaction (PCR) is a primer extension reaction thatprovides a method for amplifying specific nucleic acids in vitro.Generally, in PCR, the reaction solution is maintained for a shortperiod at each of three temperatures, 96° C., 60° C. and 72° C., toallow strand separation or denaturation, annealing, and chain extension,respectively. These three temperature stages are repeated for 30 or 40cycles with the use of an automated thermo-cycler that can heat or coolthe tube containing the reaction mixture very rapidly. By repeating thePCR cycle, a million-fold copies of a DNA sample can be produced in asingle enzymatic reaction mixture within a matter of hours, enablingresearchers to determine the size and sequence of target DNA. This DNAamplification technique has been used for cloning and other molecularbiological manipulations. Further discussion of PCR is provided inMullis, et al., Methods Enzymol. (1987), and Saiki, et al., Science(1985).

One PCR-based technique that is useful is quantitative real-time PCR(qPCR). Briefly, the mechanism of qPCR is based on PCR amplification ofa target DNA in an exponential manner. By running a PCR reaction andmeasuring the total number of DNA copies at given points during thecourse of the amplification reaction, one can retroactively calculatethe amount of starting DNA material.

Fluorescence-based DNA detection is a generally sensitive, versatile,and convenient detection method that is used in qPCR. There are twotypes of fluorescent reagents used in qPCR. The first type is based onoligonucleotides labeled with one or more fluorescent dyes, or with acombination of a fluorescent dye and a quencher dye. These labeledoligonucleotides release fluorescence either upon hybridization to atarget sequence, or upon cleavage of the oligonucleotides followinghybridization in a manner proportional to the amount of DNA present. Themechanism and the use of the oligo-based fluorescent reagents have beendescribed in various patents and publications. See, for example,Holland, et al., Proc. Natl. Acad. Sci. USA (1991); Lee, et al., NucleicAcids Res. (1993); and U.S. Pat. Nos. 5,210,015, 5,538,848, 6,258,569,5,691,146, 5,925,517, 5,118,801, 5,312,728, and 6,635,427. Althougholigo-based fluorescent reagents for qPCR have the advantage of beinghighly specific toward a target sequence, they are very complex indesign and consequently expensive to use. The second type of fluorescentreagents used in qPCR is based on DNA-binding fluorescent dyes, whichare commonly referred to as fluorescent nucleic acid dyes or stains.Because fluorescent nucleic acid dyes are relatively simple molecules,they are easy to manufacture and thus inexpensive to use. Theirapplication in qPCR is useful for routine genetic detection in researchlabs.

Not all commonly available fluorescent nucleic acid stains can be usedfor qPCR. Ideally, a fluorescent nucleic acid dye should meet certaincriteria for it to be suitable for qPCR use. First, it should bechemically stable during PCR and storage. Since PCR is carried out athigh temperature, the dye should be thermo-stable. Additionally, sincethe pH of the Tris buffer used for PCR can vary considerably fromalkaline (pH 8.5) at low temperature (4° C.) to neutral or slightlyacidic at high temperature, the dye should be resistant to acid- orbase-assisted decomposition. Second, the dye, when present in the PCRsolution, should not inhibit the PCR process. Third, the dye should benon-fluorescent or minimally fluorescent in the absence of DNA, andshould become highly fluorescent in the presence of DNA. Fourth, the dyeshould have absorption and emission wavelengths that are compatible withexisting instruments, which are normally equipped with optical channelsoptimized for common fluorescent dyes, such as FAM, JOE, VIC (AppliedBiosystems, Foster City, Calif.), TAMRA, ROX, Texas Red, Cy3, and Cy5,for example. Fifth, the dye should bind with DNA with little or nosequence preference. Sixth, the DNA-dye complexes should havefluorescence intensities that are linearly related to the amount of DNApresent.

Given the foregoing criteria, it is not surprising that very few nucleicacid-binding dyes can be used for qPCR. Ethidium bromide (EB) is a DNAdye that has been used to demonstrate the feasibility of using a simpledye for qPCR. Higuchi, et al., Bio-Technol. 10(4), 413 (1992). However,EB suffers from problems of low sensitivity and undesirable wavelengths.A widely used dye for qPCR is SYBR Green I from Molecular Probes, Inc.(Eugene, Oreg. (OR)). Wittwer, et al., Biotechniques 22(1), 130 (1997).SYBR Green I is a cyclically substituted asymmetric cyanine dye. Zipper,et al., Nucleic Acids Res. 32 (12), e103 (2004); and U.S. Pat. Nos.5,436,134 and 5,658,751. The advantages of SYBR Green I are that it hasexcitation and emission wavelengths very closely matching those of FAM,with which most of the instruments are compatible, and that it is highlyfluorescent when bound to DNA. Recently, a DNA dye called LC Green wasused for qPCR, although the structure of the dye was not disclosed.Although the LC Green dye appears to have desirable wavelengths matchingthe commonly used FAM optical channel in most of the PCR instruments, itis much less sensitive than SYBR Green I. More recently, a DNA minorgroove-binder called BEBO and a related dye called BOXTO, both of whichare asymmetric cyanine dyes, have been reported for use in qPCR.Bengtsson, et al., Nucleic Acids Res. 31 (8), e45 (2003); and U.S.Patent Application Publication No. 2004/0132046. Like LC Green, bothBEBO and BOXTO significantly lag behind SYBR Green I in terms ofsensitivity.

Although SYBR Green I has been widely used DNA dye for qPCR, it still islacking in several respects. For one, SYBR Green I has an inhibitoryeffect on the PCR process, which limits the maximum signal strength onecan achieve by increasing dye concentration. The fluorescent signalstrength of qPCR using SYBR Green I is initially proportional to the dyeconcentration until the dye concentration reaches a point where the dyestarts to inhibit the PCR process significantly. A further increase indye concentration will actually lower the signal strength or increasethe cycle number (Ct) because of reduced DNA amplification. For another,SYBR Green I is chemically unstable under alkaline conditions, such asthe alkaline condition of the PCR buffer when stored at low temperature.It has been reported that SYBR Green I stored in Tris buffer at 4° C.decomposes significantly over the course of a few days and that the dyedecomposition products are apparently potent inhibitors. Karsai, et al.,BioTechniques 32(4), 790 (2002). For yet another, SYBR Green I providesonly one fluorescence color. Many commercially available fluorescencedetection instruments have multiple optical channels (the FAM opticalchannel and additional other optical channels) and are thus capable ofdetecting multiple fluorescence colors.

Development of fluorescent dyes or the making or the use thereof isdesirable.

SUMMARY

A method of producing or designing a fluorescent dye suitable for usefulapplication, such as in a qPCR process, for example, is provided. Themethod involves covalently linking two or three monomeric dyes via abridge that may be flexible and substantially neutral (for example,neutral or slightly charged). A method of producing or designing a dye,as provided herein, may allow for the development of a fluorescentnucleic acid dye that has a wavelength and/or other spectral propertythat heretofore could not be obtained.

A fluorescent dye suitable for useful application, such as thatdescribed above, for example, is provided. A dimeric or trimeric dye,which may be produced according to a method described herein, may form ahairpin structure, which, it is believed, may enable the dye to stainnucleic acids via a release-on-demand mechanism, as further describedherein. A dye described herein may have at least one feature or all ofthe following features: relatively low “fluorescence background”(fluorescence in the absence of nucleic acids), if any, and ideally, nofluorescence background; relatively low PCR inhibition, and ideally, noPCR inhibition; relatively high fluorescent signal strength; andrelative high stability. The dye may be better as to at least one ofthese features, or as to all of these features, than an existing dye,such as SYBR Green I, merely by way of example. A dye described hereinmay have a property, such as a wavelength and/or another spectralproperty, for example, that heretofore could not be obtained.

Dimeric and/or trimeric nucleic acid dyes or stains that are capable ofintramolecular dimer formation, or the formation of a hairpin structure,are provided. It is believed that a hairpin-shaped dye maynon-fluorescent or minimally fluorescent by itself, but may becomehighly fluorescent in the presence of nucleic acids. It is believed thatnucleic acid binding of the dye may occur via an intermediate statewherein the dye forms, in part, an open random conformation. It isfurther believed that this open random conformation of the dye may existin a small quantity and in equilibrium with the hairpin state. It isbelieved that as the amount of nucleic acids increases, an equilibriumshift from the hairpin state toward the nucleic acid-bound state of thedye may occur, such that the strength of the resulting fluorescencesignal may be substantially linearly proportional to the amount ofnucleic acids present.

The above-described mechanism, which may be referred to as arelease-on-demand mechanism of DNA staining, may be desirable forvarious applications, such as quantitative, real-time PCR (qPCR), forexample. Merely by way of explanation, it is believed that the formationof the hairpin structure may render the “effective dye concentration”low, such that a dye described herein may interfere very little with thePCR process. Thus, as compared with previous dyes, such as SYBR Green I,for example, a higher concentration of a dye described herein may beused in qPCR. This higher concentration of dye may increase DNAdetection sensitivity, perhaps significantly.

A method of determining nucleic acid formation or a lack thereof in asample is provided. The sample may or may not comprise a target nucleicacid. Such a method may comprise providing a test solution comprisingthe sample and a fluorescent nucleic acid dye, where the fluorescentnucleic acid dye has the formula:

wherein BRIDGE is a substantially aliphatic, substantially neutrallinker comprising from about 8 to about 150 non-hydrogen atoms; Q₁ is adye constituent selected from a fluorescent nucleic acid dyeconstituent, a non-fluorescent nucleic acid dye constituent, afluorescent non-nucleic acid dye constituent, and a non-fluorescentnon-nucleic acid dye constituent; Q₂ is a dye constituent selected froma fluorescent nucleic acid dye constituent, a non-fluorescent nucleicacid dye constituent, a fluorescent non-nucleic acid dye constituent,and a non-fluorescent non-nucleic acid dye constituent. The dyeconstituents may be any of suitable dye constituents, such as thosedescribed herein, for example. Merely by way of example, the fluorescentnucleic acid dye constituent may be selected from an acridine dye, anasymmetric cyanine dye, a symmetric cyanine dye, a phenanthridinium dye,and a pyronin dye, and a styryl dye. At least one dye constituent of theQ₁ dye constituent and the Q₂ dye constituent is a reporter dyeconstituent, and at least one dye constituent of the Q₁ dye constituentand the Q₂ dye constituent is a fluorescent nucleic acid dye constituentor a non-fluorescent nucleic acid dye constituent. The reporter dyeconstituent and the fluorescent nucleic acid dye constituent may or maynot be the same. The method may comprise performing a process using thetest solution that would be sufficient for amplification of the targetnucleic acid should the sample comprise the target nucleic acid. Merelyby way of example, the process may be a PCR process, such as a real-timePCR process, for example. The method may comprise illuminating the testsolution with light at a wavelength sufficient for absorption by thereporter dye constituent and determining fluorescent emission or a lackthereof.

Another method of determining nucleic acid formation or a lack thereofin a sample is provided. The sample may or may not comprise a targetnucleic acid. Such a method method may comprise providing a testsolution comprising the sample and a fluorescent nucleic acid dye, wherethe fluorescent nucleic acid dye has the formula:

wherein BRIDGE is a substantially aliphatic, substantially neutrallinker comprising from about 15 to about 150 non-hydrogen atoms; Q₁ is adye constituent selected from a fluorescent nucleic acid dyeconstituent, a non-fluorescent nucleic acid dye constituent, afluorescent non-nucleic acid dye constituent, and a non-fluorescentnon-nucleic acid dye constituent; Q₂ is a dye constituent selected froma fluorescent nucleic acid dye constituent, a non-fluorescent nucleicacid dye constituent, a fluorescent non-nucleic acid dye constituent,and a non-fluorescent non-nucleic acid dye constituent; Q₃ is a dyeconstituent selected from a fluorescent nucleic acid dye constituent, anon-fluorescent nucleic acid dye constituent, a fluorescent non-nucleicacid dye constituent, and a non-fluorescent non-nucleic acid dyeconstituent. The dye constituents may be any suitable dye constituents,such as those described herein, for example. Merely by way of example,the fluorescent nucleic acid dye constituent may be selected from anacridine dye, an asymmetric cyanine dye, a symmetric cyanine dye, aphenanthridinium dye, and a pyronin dye, and a styryl dye. At least onedye constituent of the Q₁ dye constituent, the Q₂ dye constituent, andthe Q₃ dye constituent is a reporter dye constituent, and at least onedye constituent of the Q₁ dye constituent, the Q₂ dye constituent, andthe Q₃ dye constituent is a fluorescent nucleic acid dye constituent ora non-fluorescent nucleic acid dye constituent. The reporter dyeconstituent and the fluorescent nucleic acid dye constituent may or maynot be the same. The method may comprise performing a process using thetest solution that would be sufficient for amplification of the targetnucleic acid should the sample comprise the target nucleic acid. Merelyby way of example, the process may be a PCR process, such as a real-timePCR process, for example. The method may comprise illuminating the testsolution with light at a wavelength sufficient for absorption by thereporter dye constituent and determining fluorescent emission or a lackthereof.

In the formulas provided above, BRIDGE may have the formula set forthdirectly below.-L₁-[A¹-(CH₂)_(α)-]_(a)[A²-(CH₂)_(β)-]_(b)[A³-(CH₂)_(γ)-]_(c)[A⁴-(CH₂)_(δ)-]_(d)[A⁵-(CH₂)_(ε)-]_(e)[A⁶-(CH₂)_(ζ)-]_(f)[A⁷-(CH₂)_(η)-]_(g)[A⁸-(CH₂)_(θ)-]_(h)[A⁹-(CH₂)_(τ)-]₁-A¹⁰-L₂-In this formula, each of L₁ and L₂, independently, is a moietycomprising a single bond; a polymethylene unit having 1 carbon to about12 carbons, inclusive, optionally comprising at least one hetero atomselected from N, O and S; or an aryl optionally comprising at least onehetero atom selected from N, O and S; each of A¹, A², A³, A⁴, A⁵, A⁶,A⁷, A⁸, A⁹, and A¹⁰, independently, is anucleic-acid-binding-enhancing-group (NABEG); a branched alkyloptionally comprising at least one hetero atom selected from N, O and S;or at least one saturated 5- or 6-membered ring, optionally comprisingat least one hetero atom selected from N, O and S; each of α, β, γ, δ,ε, ζ, η, θ, and τ, independently, is zero or an integer from 1 to about20, inclusive; and each of a, b, c, d, e, f, g, h, and i, independently,is zero or an integer from 1 to about 20, inclusive. BRIDGE may comprisea suitable number of non-hydrogen atoms, such as from about 8 to about100 or about 150, inclusive, about 12 to about 60, inclusive, or about15 to about 40, inclusive, merely by way of example. BRIDGE may compriseup to one positive charge, merely by way of example. BRIDGE may be anysuitable linker molecule, such as any described herein, for example. Inone example, BRIDGE has the formula:—(CH₂)_(x)—C(═O)NH—(CH₂)_(α)—[O—(CH₂)_(β)]_(b)—[O—(CH₂)_(γ)]_(c)—NH(O═C)—(CH₂)_(x)—wherein each x, independently, is an integer selected from 1 to 11,inclusive; α is an integer selected from 2 to about 20, inclusive; eachof β and γ, independently, is 2 or 3; b is zero or an integer from 1 toabout 20, inclusive; and c is zero or 1.

Compositions associated with the above-described methods are alsoprovided. Merely by way of example, a dye of any of the structuresprovided directly below is provided.

In the dye structures above, Ψ represents an anion, such as a iodide ora chlorine anion, merely by way of example.

A composition having the formula set forth directly below is alsoprovided.

In this formula, BRIDGE is a substantially aliphatic linker comprisingfrom about 15 to about 150 non-hydrogen atoms and up to one positivecharge; Q₁ is a fluorescent nucleic acid dye constituent; Q₂ is afluorescent nucleic acid dye constituent; and R_(r) is a reactive groupor a functional group. The reactive group or functional group may be anyof suitable such groups, such as those described herein, for example.The composition may be any suitable composition, such as any of thosedescribed herein, for example. A method of using the composition, ordye, may comprise conjugating the composition to a substrate molecule,such as a substrate molecule selected from a nucleotide, anoligonucleotide, a peptide, a protein, a hapten, a drug, amicroparticle, a synthetic polymer, a natural polymer, a biologicalcell, a virus, and a molecule of a solid surface.

A method of preparing a sample that may or may not comprise nucleic acidis also provided. The method may comprise providing a combination of thesample and a composition, or dye, such as those described herein,wherein if nucleic acid is present in the sample, a nucleic acid-dyecomplex is formed. The method may further comprise incubating thecombination. A method of determining presence or absence of nucleic acidin a sample, is also provided. The method may comprise providing acombination of the sample and a composition, or dye, such as thosedescribed herein, wherein if nucleic acid is present in the sample, anucleic acid-dye complex is formed; illuminating the combination withlight at a wavelength sufficient such that if a nucleic acid-complex isformed the light is absorbed thereby; and determining fluorescentemission or a lack thereof. A kit for determining nucleic acid formationor a lack thereof in a sample is also provided. The kit may comprise atleast one composition sufficient for amplification of the target nucleicacid in the sample should the sample comprise the target nucleic acid,and a composition, or dye, such as those described herein.

These and various other aspects, features, and embodiments are furtherdescribed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

A detailed description of various aspects, features and embodiments isprovided herein with reference to the accompanying drawings, which arebriefly described below. The drawings are illustrative and are notnecessarily drawn to scale. The drawings illustrate various aspects orfeatures and may illustrate one or more embodiment(s) or example(s) inwhole or in part. A reference numeral, letter, and/or symbol that isused in one drawing to refer to a particular element or feature may beused in another drawing to refer to a like element or feature.

FIG. 1 is a schematic illustration of DNA binding via arelease-on-demand mechanism, in which three conformation states of thedye are in substantial equilibrium.

FIG. 2 is a graphical representation of normalized absorbance versuswavelength (nm), or normalized absorption spectra, of a) a dimeric dye,AOAO-7 (∘), and b) a monomeric AO dye, DMAO (Δ), in PBS buffer.

FIG. 3 is a graphical representation of normalized absorbance versuswavelength (nm), or normalized absorption spectra, of a) a dimeric dye,AOAO-7 (●), and b) a monomeric AO dye, DMAO (▴), in a buffer and in thepresence of DNA.

FIG. 4 is a graphical representation of relative fluorescence versuswavelength (nm), or fluorescence emission spectra, of DMAO (Δ) andAOAO-7 (∘) in PBS buffer before DNA addition, a) and c), respectively,and DMAO (▴) and AOAO-7 (●) in PBS buffer after DNA addition, b) and d),respectively.

FIG. 5 is a graphical representation of normalized absorbance versuswavelength (nm), or normalized absorption spectra, of a) TOTO-1(prepared according to U.S. Patent No. 5,582,977) (darker line), and b)TOTO-3 (lighter line), in a buffer.

FIG. 6 is a graphical representation of normalized absorbance versuswavelength (nm), or normalized absorption spectra, of a) TOTO-1according to U.S. Pat. No. 5,582,977) (darker line), and b) TOTO-3(lighter line), in a buffer and in the presence of DNA.

FIG. 7 includes a graphical representation of relative fluorescenceversus DNA concentration (μg/mL), or a titration, of single-stranded DNA(⋄), and double-stranded DNA (♦), in solution and in the presence ofAOAO-12 (at 0.2 μM). FIG. 7 also includes an inset graphicalrepresentation of relative fluorescence versus DNA concentration thatshows a substantially linear relationship between the two.

FIG. 8 is a graphical representation of relative fluorescence versuscycle number (Ct), or PCR amplification, using SYBR Green I at a lowerdye concentration (Δ) and a higher dye concentration (▴), and AOAO-12 ata lower dye concentration (□) and a higher dye concentration (▪), whereCt generally refers to the cycle number at which the fluorescence signalreaches an arbitrary threshold and, in a PCR amplification plot,generally corresponds to where the fluorescence signal just begins torise from the baseline. For each dye, relative dye concentration wasmeasured in optical density (OD) at the absorption maximum of the dye inPBS buffer, with the dye concentration of 1× and 2× AOAO-12 at 471 nmcorresponding to an OD of 0.1 and 0.2, respectively, and the dyeconcentration of 0.5× and 1× SYBR Green I at 495 nm corresponding to anOD of 0.025 and 0.05, respectively.

FIG. 9 includes a graphical representation of relative fluorescenceversus cycle number (Ct), or PCR amplification, using AOAO-12 (□), andSYBR Green I (Δ), as the fluorescent probe, each at optimalconcentrations. For each dye, relative dye concentration was measured inoptical density (OD) at the absorption maximum of the dye in PBS buffer,with the dye concentration of AOAO-12 at 471 nm corresponding to an ODof 0.4 and the dye concentration of SYBR Green I at 495 nm correspondingto an OD of 0.025. FIG. 9 also includes an inset graphicalrepresentation of Ct versus log of DNA sample copy number, using AOAO-12(▪) and SYBR Green I (▴), respectively, that in each case, shows asubstantially linear relationship between the two.

FIG. 10 is a schematic illustration of possible combinations A, B, C, D,and E, of monomeric dyes, Q₁ and Q₂, to form a dimeric dye. CombinationA comprises two identical reporter fluorescent nucleic acid dyes or tworeporter fluorescent nucleic acid dyes of similar spectra. Combination Bcomprises one reporter fluorescent nucleic acid dye and one non-reporterDNA-binding molecule. Combination C comprises one non-reporterDNA-binding molecule and one reporter non-DNA-binding dye. Combination Dcomprises one reporter fluorescent nucleic acid dye and one non-reporternon-fluorescent non-nucleic acid dye. Combination E comprises onereporter fluorescent nucleic acid dye and one reporter fluorescentnon-nucleic acid dye.

FIG. 11 is a graphical representation of fluorescence intensity versuscycle number (Ct), or PCR amplification, using TO monomeric dye (∘),TOTO-1 monomeric dye (Δ), and TOTO-12 dimeric dye (□) (Compound No. 24,Table 2).

FIG. 12 is a graphical representation of absorbance versus wavelength(nm), or absorption spectra, of heterodimeric dye AORO-7 in PBS buffer(∘), and of heterodimeric dye AORO-7 in PBS buffer and in the presenceof DNA (+).

FIG. 13 is a graphical representation of arbitrary fluorescenceintensity versus wavelength (nm), or emission spectra, of theheterodimeric dye AORO-7, with excitation either at 500 nm (solid,darker line) or at about 560 nm (broken, lighter line), recordedseparately.

FIG. 14 includes a graphical representation of relative fluorescenceversus cycle number (Ct), or PCR amplification, using the heterodimericdye AORO-7. FIG. 14 also includes an inset graphical representation, ormelting curve plot, of relative fluorescence signal versus temperature(° C.).

FIG. 15 is a graphical representation, or melting curve plot, ofrelative fluorescence change versus temperature (° C.), A) using AOAO-12(dashed lines) monitoring, and B) using SYBR Green I (solid lines)monitoring, of four amplicons, TBP (∘), SDHA (Δ), RPL4 (⋄), and HMBS(□).

FIG. 16 is a graphical representation of relative fluorescence monitoredat 60° C. versus minutes at 96° C., or thermo-stability at 96° C., ofAOAO-12.

DESCRIPTION

Fluorescent dyes or stains that may be useful in various applications,such as nucleic acid detection, for example, are described herein. Suchdyes may be dimeric or trimeric nucleic acid dyes, for example, thathave low background fluorescence in the absence of nucleic acids, butbecome highly fluorescent in the presence of nucleic acids. Dimeric andtrimeric nucleic acid dyes may be useful in various applications, suchas nucleic acid detection, for example, as described herein. Methodsassociated with the preparation and use of fluorescent dyes or stainsare also described herein, as are useful systems, or kits, that comprisefluorescent dyes or stains.

Herein, it will be understood that a word appearing in the singularencompasses its plural counterpart, and a word appearing in the pluralencompasses its singular counterpart, unless implicitly or explicitlyunderstood or stated otherwise. Further, it will be understood that forany given component described herein, any of the possible candidates oralternatives listed for that component, may generally be usedindividually or in combination with one another, unless implicitly orexplicitly understood or stated otherwise. Additionally, it will beunderstood that any list of such candidates or alternatives, is merelyillustrative, not limiting, unless implicitly or explicitly understoodor stated otherwise. Still further, it will be understood that anyfigure or number presented herein is approximate, and that any numericalrange includes the minimum number and the maximum number defining therange, whether or not the term “inclusive” or the like appears, unlessimplicitly or explicitly understood or stated otherwise. Additionally,it will be understood that any permissive, open, or open-ended languageencompasses any relatively permissive to restrictive language, open toclosed language, or open-ended to closed-ended language, respectively,unless implicitly or explicitly understood or stated otherwise. Merelyby way of example, the word “comprising” may encompass “comprising”-,“consisting essentially of”-, and/or “consisting of”-type language.

Various terms are generally described below or used herein to facilitateunderstanding. It will be understood that a corresponding generaldescription of these various terms applies to corresponding linguisticor grammatical variations or forms of these various terms. It will alsobe understood that the general description of any term below may notapply or may not fully apply when the term is used in a non-general ormore specific manner. It will also be understood that the terminologyused or the description provided herein, such as in relation to variousembodiments, for example, is not limiting. It will further be understoodthat embodiments described herein or applications described herein, arenot limiting, as such may vary.

Generally, the terms “stain” and “dye” may be used interchangeably andrefer to an aromatic molecule capable of absorbing light in the spectralrange of from about 250 nm to about 1,200 nm. Generally, the term “dye”may refer to a fluorescent dye, a non-fluorescent dye, or both.Generally, the term “fluorescent dye” refers to a dye capable ofemitting light when excited by another light of appropriate wavelength.

Generally, the term “fluorescence quencher” refers to a molecule capableof quenching the fluorescence of another fluorescent molecule.Fluorescence quenching can occur via at least one of the three ways. Thefirst type of fluorescence quenching occurs via fluorescence resonanceenergy transfer (FRET) (Förster, Ann. Phys. (1948); and Stryer, et al.,Proc. Natl. Acad. Sci. (1967)), wherein a quencher absorbs the emissionlight from a fluorescent molecule. The absorption peak of a FRETquencher usually has to have significant overlap with the emission peakof a fluorescent dye for the FRET quencher to be an efficientfluorescent quencher. A FRET quencher is typically a non-fluorescentdye, but can also be a fluorescent dye. When a quencher is a fluorescentdye, only the absorption property of the dye is utilized. A second typeof fluorescence quenching occurs via photo-induced electron transfer(PET), wherein the quencher is an electron-rich molecule that quenchesthe fluorescence of a fluorescent molecule by transferring an electronto the electronically excited dye. A third type of fluorescencequenching occurs via dye aggregation, such as H-dimer formation, whereintwo or more dye molecules are in physical contact with one another,thereby dissipating the electronic energy into the vibrational modes ofthe molecules. This type of contact fluorescence quenching can occurbetween two identical fluorescent dyes, or between two differentfluorescent dyes, or between a fluorescent dye and a FRET quencher, orbetween a fluorescent dye and a PET quencher. Other types offluorescence quenchers, though not used as commonly, include stable freeradical compounds and certain heavy metal complexes.

Generally, the term “nucleic acid” refers to double-stranded DNA(dsDNA), single-stranded DNA (ssDNA), double-stranded RNA (dsRNA),single-stranded RNA (ssRNA), and/or derivatives thereof. A nucleic acidmay be natural or synthetic.

Generally, the term “fluorescent nucleic acid stain” or “fluorescentnucleic acid dye” refers to a dye capable of binding to a nucleic acidto form a fluorescent dye-nucleic acid complex. A fluorescent nucleicacid dye is typically non-fluorescent or weakly fluorescent by itself,but becomes highly fluorescent upon nucleic acid binding. Generally, theterm “non-fluorescent, nucleic acid-binding molecule” refers to anucleic acid-binding molecule that may or may not be a dye and that doesnot become fluorescent upon binding to nucleic acid. Generally, the term“fluorescent DNA dye” refers to a dye that becomes fluorescent uponbinding to DNA. Generally, the term “fluorescent, non-nucleic acid dye”refers to a fluorescent dye that does not bind to nucleic acid.Generally, the term “non-fluorescent, non-nucleic acid dye” refers to adye that is neither fluorescent nor nucleic acid-binding. Such a dye iscommonly called a fluorescence quencher. Frequently, a fluorescencequencher is used to form a FRET pair with a fluorescent dye. Generally,the term “reporter dye” refers to a fluorescent dye whose emittedfluorescence contributes to the final detected fluorescence signal.

Generally, the term “polymerase chain reaction” or “PCR” refers to atechnique for amplifying the amount of DNA. Generally, the term“quantitative, real-time PCR” or “qPCR” refers to a technique to monitorthe growing amount of DNA in the course of a PCR.

In general, fluorescent nucleic acid dyes can be classified into twomajor classes: intercalators and minor groove-binders. Generally,fluorescent intercalators are dyes that bind to double-stranded DNA ordouble-stranded RNA by inserting themselves in between a neighboringbase pair. Generally, minor groove-binders are dyes that bind to theminor groove of double-stranded DNA. There are still other dyes that maybind to nucleic acids via multiple modes, including electrostaticinteraction between a positively charged dye and the negatively chargednucleic acid.

Although a variety of fluorescent nucleic acid dyes have becomecommercially available, and methods for improving the dyes for non-qPCRuses have been developed, not all nucleic acid stains are suitable forqPCR application. Additionally, little is known as to what structuralelements are required for a good qPCR dye.

In general, from a performance point of view, an ideal dye for qPCRshould meet various criteria, as now described. The dye should bethermally stable at high temperature (from about 60° C. to about 96° C.)in a PCR buffer and hydrolytically stable at low temperature (from about−20° C. to about 4° C.) when the media becomes alkaline. The dye shouldnot inhibit the PCR process, this generally being the most importantcriteria, as in the most severe cases of PCR inhibition, the PCR processmay not even start, and in milder cases, the Ct number may be delayed,or only a very low dye concentration may be used, such that thefluorescence signal is limited. The dye should be non-fluorescent orminimally fluorescent in the absence of DNA, but become highlyfluorescent in the presence of DNA. The absorption and emissionwavelengths of the dye should be compatible with instruments used inconnection with qPCR, such as the existing instruments previouslydescribed. The DNA binding of the dye should have little or no sequencepreference. The fluorescence intensity of the DNA-dye complexes shouldbe linearly related to the amount of DNA present. A dye described hereinmay or may not meet one or more of the above-described criteria.

A method for designing a fluorescent nucleic acid dye, such as onesuitable for qPCR, for example, is provided. The method comprisescovalently linking two or three monomeric dyes with a suitable linker toform a dimeric dye or a trimeric dye. A dye described herein, when insolution, may assume a predominantly hairpin-like conformation due tointramolecular dimer formation. This hairpin-like conformation or stateof the dye is inactive with respect to nucleic acids, or incapable ofinteracting with nucleic acids. It is believed that the dye, when insolution and in the presence of nucleic acids, also assumes an openrandom conformation or state, which exists in small quantity and insubstantial equilibrium with the hairpin conformation. The open randomconformation or state of the dye is active with respect to nucleicacids, or capable of interacting or binding with nucleic acids. It isbelieved that when the dye is in the presence of an increasing amount ofnucleic acids, an equilibrium shift from the hairpin state toward theintermediate, open random state, or DNA-binding state, occurs. It isbelieved that this mechanism, sometimes referred to as a“release-on-demand DNA-binding mechanism,” reduces PCR inhibition thatmay otherwise be associated with the dye. As a consequence, the dye maybe used in PCR processes at a higher concentration than might otherwisebe possible, and thus, may provide for greater nucleic acid detectionsensitivity than might otherwise be possible. The reduction in PCRinhibition may be dramatic, and the increase in nucleic acid detectionsensitivity may be significant.

A dimeric dye or trimeric dye described herein may posses any number ofdesirable characteristics. By way of example, such a dye may have abackground fluorescence that is reduced relative to that of itsmonomeric dye constituents. Relatively low background fluorescencegenerally corresponds to relatively enhanced nucleic acid detectionsensitivity. Thus, such a dye is generally associated with enhancednucleic acid detection sensitivity. Further by way of example, a dyedescribed herein may be more thermally and/or hydrolytically stable thanSYBR Green I. Still further by way of example, a dye described hereinmay have absorption and emission wavelengths other than those associatedwith existing qPCR dyes.

A fluorescent dimeric nucleic acid dye may have the general structure(Structure 1) set forth directly below.

In Structure 1, independently, each dye of dye Q₁ and dye Q₂ is selectedfrom a fluorescent nucleic acid dye, a non-fluorescent nucleic acid dye,a fluorescent non-nucleic acid dye, and a non-fluorescent non-nucleicacid dye. Q₁ and Q₂ may be selected and combined in a manner toencourage or to ensure desired properties of the resulting dimeric dye.At least one dye of dye Q₁ and dye Q₂ is a reporter dye. Further, atleast one dye of dye Q₁ and dye Q₂ is a fluorescent nucleic acid dye ora non-fluorescent nucleic acid dye. The reporter dye and fluorescentnucleic acid dye may be the same or different. BRIDGE may be positivelycharged to a relatively limited extent or substantially neutral incharge, and may be a substantially flexible constituent that facilitatesintramolecular dimer formation to produce the dimeric dye.

A fluorescent trimeric nucleic acid dye may have the general structure(Structure 2) set forth directly below.

In Structure 2, independently, each dye of dye Q₁, dye Q₂, and dye Q₃ isselected from a fluorescent nucleic acid dye, a non-fluorescent nucleicacid dye, a fluorescent non-nucleic acid dye, and a non-fluorescentnon-nucleic acid dyes. Q₁, Q₂, and Q₃ may be selected and combined in amanner to encourage or to ensure desired properties of the resultingtrimeric dye. At least one dye of dye Q₁, dye Q₂, and dye Q₃ is areporter dye. Further, at least one dye of dye Q₁, dye Q₂, and dye Q₃ isa fluorescent nucleic acid dye or non-fluorescent nucleic acid dye. Thereporter dye and fluorescent nucleic acid dye may be the same ordifferent. BRIDGE may be positively charged to a relatively limitedextent or substantially neutral in charge, and may be a substantiallyflexible constituent that facilitates intramolecular dimer formation toproduce the trimeric dye.

A fluorescent nucleic acid dye may have the general structure (Structure3) set forth directly below.

In Structure 3, independently, each dye of dye Q₁, dye Q₂, may be asdescribed above in relation to Structure 1 and Structure 2; BRIDGE maybe a substantially aliphatic linker, as previously described; and R_(r)may be a reactive group or a functional group. Merely by way of example,Q₁ may be a fluorescent nucleic acid dye constituent; Q₂ may be afluorescent nucleic acid dye constituent; BRIDGE may be a substantiallyaliphatic linker comprising from about 15 to about 150 non-hydrogenatoms and up to one positive charge; and R_(r) may be a reactive groupor a functional group, as described herein.

BRIDGE

BRIDGE may be a substantially flexible linker molecule, having no morethan one positive charge. BRIDGE may be a substantially neutral andsubstantially flexible linker molecule. The constituents of BRIDGE maybe selected to achieve such a limited positive charge or such asubstantial neutrality. The property of substantial neutrality, whichincludes actual neutrality, is discussed further below. The property ofsubstantial flexibility is generally related to the substantiallyaliphatic nature, which includes actual aliphatic nature, of BRIDGE.This substantial aliphatic nature generally refers to thenon-aromaticity of BRIDGE, or non-rigidity of BRIDGE.

In Structure 1, BRIDGE is covalently attached to Q₁ and Q₂. In the caseof dimeric dyes, BRIDGE may have from about 8 to about 150 non-hydrogenatoms, from about 8 to about 100 non-hydrogen atoms, from about 12 toabout 60 non-hydrogen atoms, or from about 15 or about 20 to about 40 orabout 50 non-hydrogen atoms, for example. In Structure 2, BRIDGE iscovalently attached to Q₁, Q₂ and Q₃. In the case of trimeric dyes,BRIDGE may have from about 15 to about 150 non-hydrogen atoms, fromabout 20 to about 150 non-hydrogen atoms, from about 20 to about 100non-hydrogen atoms, or from about 30 to about 70 non-hydrogen atoms, forexample.

BRIDGE may incorporate at least one independentnucleic-acid-binding-enhancing-group (NABEG). A NABEG is a moietycapable of binding to nucleic acids in the form of electrostatic,hydrophobic, or hydrogen-bonding interactions. Merely by way of example,a NABEG may be selected from primary amines; secondary amines; tertiaryamines; ammoniums; amidines; aryl groups optionally comprising heteroatoms selected from N, O, S, and any combination thereof; moietieshaving bonds comprising hetero atoms of high electronegativity; and anycombination thereof.

Primary, secondary and tertiary amines and amidines are basic groups andtherefore are positively charged or at least partially positivelycharged at physiological pH. Ammonium groups, or quaternized nitrogengroups, are permanently positively charged. Generally speaking,positively charged or partially positively charged groups enhance thenucleic acid binding of the dye via electrostatic interaction, aproperty that may be exploited in the development of highly sensitivefluorescent nucleic acid stains. It is generally undesirable to useBRIDGE having excessive positive charges to produce a dye of the presentinvention. For example, a suitable BRIDGE of a dimeric dye or a trimericdye of the invention may comprise no more than one positive charge.BRIDGE may be a substantially flexible and neutral or substantiallyneutral linker. In this context, substantially neutrality refers toslight charge. By way of example, BRIDGE could comprise a weakly basicconstituent, such as a pyridine group or a pyrazine group, for example,such that when it is in aqueous solution, a very small amount ofpositive charges may be present. Further by way of example, in a case(optional) in which BRIDGE comprises at least one neutral NABEG, theexact amount of positive charge may be generally related to the pK_(a)of the NABEG. Generally, the higher the pK_(a) of the NABEG, the morelikely the NABEG is protonated and thus, positively charged. By way ofexample, a suitable weakly basic NABEG group may have a pK_(a) of about11 or less, about 8 or less, or about 7 or less.

There may be a tendency to form an intramolecular dimer, primarilyH-dimer, which may be a particularly useful property in the nucleic aciddye produced. For example, in the case of a dimeric dye describedherein, H-dimer formation may produce a hairpin-like structure, whereinH-dimer forms a stem portion of the hairpin and BRIDGE forms a curvedportion, as schematically illustrated in FIG. 1. The phenomenon ofH-dimer formation in connection with certain dyes has been described inWest, et al., J. Phys. Chem. (1965); Rohatgi, et al., J. Phys. Chem.(1966); Rohatgi, et al., Chem. Phys. Lett. (1971); and Khairutdinov, etal., J. Phys. Chem. (1997). Formation of an intramolecular H-dimer maybe facilitated when BRIDGE is a flexible and neutral or substantiallyneutral hydrocarbon linker, optionally comprising one or more neutralNABEG(s).

H-dimer formation may be characterized by a large blue shift of the dyeabsorption spectrum. By way of example, the absorption spectra of amonomeric dye AO (acridine orange) and a related dimeric dye, AOAO-7,that forms an intramolecular dimer, are shown in FIG. 2. The 471 nm peakassociated with the AOAO-7 dimer indicates intramolecular H-dimerformation. The absorption spectra of both the monomer and the dimerbecome similar once DNA-binding occurs, indicating the opening up of thehairpin structure. By way of example, as shown in FIG. 3, thedisappearance of the 471 nm peak from AOAO-7 dimer indicates the openingup of the hairpin structure upon DNA binding.

H-dimer formation in a dye described herein may be associated with twomajor benefits. One of the major benefits is a reduction, sometimesdramatic, in background fluorescence, coupled with a substantialincrease in fluorescence upon DNA-binding, as demonstrated by a largegain in the fluorescence signal. This benefit may be appreciated bycomparing the fluorescence spectra of a monomeric acridine orange dye,DMAO, and a dimeric acridine orange dye, AOAO-7, in the absence andpresence of DNA. For example, as shown in FIG. 4, relative to themonomeric DMAO dye, the dimeric AOAO-7 dye is associated with lowerbackground fluorescence and higher fluorescence upon binding to DNA.

Intramolecular dimer-associated fluorescence quenching may be soefficient that a dye described herein may be constructed from at leastone monomeric dye that is not normally considered to be very desirable,such as at least one monomeric dye that has high backgroundfluorescence, for example. An example of this is shown in FIG. 4, whichfeatures acridine orange (AO) and a dimer thereof. Although AO is one ofthe earliest known nucleic acid-binding dyes and has desirablewavelengths, it has not been widely used for nucleic acid detectionbecause of its relatively high background fluorescence. As demonstratedin FIG. 4, relative to the monomeric AO dye, the dimeric dye AOAO-7 hasmuch lower background fluorescence.

H-dimer formation occurs via intramolecular, rather than intermolecular,interaction. The H-dimer formation occurs via the covalent linkage oftwo or three dyes, such as a pair of desirable monomeric dyes, forexample. The H-dimer formation may be accomplished relatively easily,without the need for an additional reagent, such as an additionalreagent that may interfere with a useful application of the dye, forexample. By way of example, a dimeric dye may be formed from a pairingof one nucleic acid-binding dye constituent and one non-nucleicacid-binding fluorescent dye constituent in solution, without the use ofan additional reagent. Further by way of example, a trimeric dye may beprepared from two nucleic acid-binding dye constituents and onenon-nucleic acid-binding fluorescent dye constituent in solution, again,without the use of an additional reagent. The H-dimer formation providesa useful way to trap the dye in a non-DNA-binding state, which has noinhibitory effect on PCR and which shifts to the open random state orthe DNA-binding state only when DNA is present. Thus, the effective dyeconcentration, or the concentration of the dye in the open random state,can be kept low, even though a high total dye concentration is used toincrease the qPCR sensitivity.

As mentioned above, H-dimer formation in a dye described herein may beassociated with another major benefit. This unexpected benefit is thatH-dimer formation in a dye may significantly reduce the inhibitoryeffect of the dye to PCR in a qPCR application. By way of example,usually, for a DNA sample of a given concentration, the fluorescentsignal from a fluorescent DNA dye is proportional to the dyeconcentration, until dye saturation. By way of example, a higher dyeconcentration is associated with a greater formation of DNA-dyecomplexes, and thus, greater, or brighter, fluorescence, until dyesaturation. Therefore, ideally, one would wish to start with a highenough dye concentration for maximal sensitivity in a qPCR application.In practice, however, all DNA dyes that have previously used for qPCRinhibit the DNA amplification process in varying degrees. Typically, ahigher concentration of such a previous dye has been associated withgreater dye inhibition of the amplification or PCR process. Thus, theconcentration of such a previous dye has usually been made to be muchlower for a qPCR application than it would be for a non-qPCRapplication.

This lowering of dye concentration in qPCR applications results in asacrifice in terms of end-point fluorescent signal strength, as may bediscerned from the following discussion of the most widely used DNA dyefor qPCR, SYBR Green I, by way of example. The concentration of SYBRGreen 1 that used in qPCR is not provided by the manufacturer, such thatprecise comparison with other dyes is not easy or routine. However, asthe concentration of a dye is linearly related to its optical densityaccording to Beer's law, the optical density characteristics of SYBRGreen 1 solution used in qPCR and another dye solution used in qPCR maybe used to at least qualitatively compare the respective concentrations.For example, a typical optical density for SYBR Green 1 solution used inqPCR is from 0.025 to 0.05, while the optical density for a solution ofDye No. 19 of Table 2 herein used in qPCR is typically from about 0.04to about 0.8, or more typically, from about 0.1 to about 0.4. The SYBRGreen I dye shows a significant inhibition effect when the dyeconcentration is increased from 0.5×, which corresponds to an opticaldensity of 0.025 at the absorption peak of 495 nm, to a 1×concentration, which corresponds to an optical density of 0.05 at thesame absorption peak. As shown in FIG. 8, while SYBR Green I at the 1×concentration has a higher end-point fluorescent signal relative to SYBRGreen I at the 0.5× concentration, the cycle number, or Ct value,associated with the 1× dye concentration is delayed. This delayed Ctvalue indicates that SYBR Green I significantly inhibits PCR at higherdye concentration. The dimeric dye, AOAO-12, exhibits little or noinhibition when the dye concentration is increased from a 1× dyeconcentration, which corresponds to an optical density of 0.1 at theabsorption peak of 471 nm, to a 2× dye concentration, which correspondsto an optical density of 0.2 at the same absorption peak. BecauseAOAO-12 shows little or no PCR inhibition, it can be used at aconcentration that is relatively high and provide a fluorescent signalthat can be several times higher than that of SYBR Green I, as shown inFIG. 9. In brief, AOAO-12 shows little or no PCR inhibition within awide concentration range, and thus can be used at a higher concentrationfor an increased fluorescent signal.

It is believed that the above-described substantial lack of PCRinhibition that may be associated with dyes described herein, such thata higher dye concentration can be used, may be explained by a“release-on-demand” mechanism that is schematically illustrated inFIG. 1. That is, it is believed that in solution, a dimeric dye existsin a dynamic equilibrium between a closed hairpin conformation and anopen random conformation, as shown in FIG. 1. In general, the hairpinconformation is much more stable than the open random conformation andis predominant. The dominance of the hairpin conformation of the dye issupported by ultraviolet/visible spectra, which show a substantial shiftof the dimer spectrum relative to the monomer spectrum. The hairpinconformation is an inactive form of the dye, while the open conformationis an active form of the dye, capable of DNA binding. When DNA ispresent, dye in the open conformation shifts to a DNA-boundconformation, more dye in the hairpin conformation shifts to the openconformation, and dye in the open conformation stays at a very lowconcentration. In other words, the majority of the dye is trapped in thenon-DNA-binding hairpin conformation and is only released to the openconformation, or the DNA-binding form, in response to greater DNApresence. This “release-on-demand” mechanism makes it possible for a dyeto be used at a relatively high concentration without adverselyaffecting the PCR process itself. Unlike a dye described herein, SYBRGreen I does not have a non-DNA-binding conformation that helps lowerthe effective concentration of the dye and thus shows a highlyconcentration-dependent PCR inhibitory effect.

The nucleic acid stains described herein are relatively simple and canbe prepared on a desirable scale, such as in amounts measured in gramsto tens of grams, for example, on a fairly routine basis. These nucleicacid stains may be used fairly universally for detection of DNAamplification and for relatively routine research applications. By wayof example, fluorescent nucleic acid dyes may be used to detect thepresence and amount of DNA in a substantially non-sequence-selectivemanner, and in a relatively universal manner.

BRIDGE may have the formula (Formula 1) set forth directly below.

Formula 1-L₁-[A¹-(CH₂)_(α)-]_(a)[A²-(CH₂)_(β)-]_(b)[A³-(CH₂)_(γ)-]_(c)[A⁴-(CH₂)_(δ)-]_(d)[A⁵-(CH₂)_(ε)-]_(e)[A⁶-(CH₂)_(ζ)-]_(f)[A⁷-(CH₂)^(η)-]_(g)[A⁸-(CH₂)_(θ)-]_(h)[A⁹-(CH₂)_(τ)-]₁-A¹⁰-L₂-

In Formula 1, each substituent of substituent L₁ and substituent L₂(each of which may be referred to as simply “L”) is part of BRIDGE. L₁is covalently bound to one dye constituent of the Q₁ dye constituent andthe Q₂ dye constituent, and L₂ is covalently bound to the dyeconstituent of the Q₁ dye constituent and the Q₂ that is other than saidone dye constituent. Independently, each of L₁ and L₂ is a moietycomprising a single bond; a polymethylene unit having 1 carbon to about12 carbons optionally comprising at least one hetero atom selected fromN, O and S; or an aryl group optionally comprising at least one heteroatom selected from N, O and S. The subscripts associated with the (CH₂)methylene units, namely, α, β, γ, δ, ε, ζ, η, θ, and τ, may be the sameor different, each independently indicating the size of the associatedmethylene unit and being zero or an integer from 1 to about 20 or from 1to about 12. The subscripts associated with the bracketed portions ofFormula 1, namely, a, b, c, d, e, f, g, h, and i, may be the same ordifferent, each independently indicating the size of the associatedbracketed portion of the formula and being zero or an integer from 1 toabout 20, or from 1 to about 10 or from 1 to about 5.

A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ may be the same ordifferent, each, independently, being anucleic-acid-binding-enhancing-group (NABEG); a branched alkyloptionally comprising at least one hetero atom selected from N, O and S;or at least one saturated 5- or 6-membered ring optionally comprising atleast one hetero atom selected from N, O and S. A¹, A², A³, A⁴, A⁵, A⁶,A⁷, A⁸, A⁹, and A¹⁰ may be such that BRIDGE comprises at most onepositive charge, or is substantially neutral, and in the latter case,each of these constituents, independently, may itself be substantiallyneutral, which includes actual neutrality. NABEGs may be selected frommoieties comprising at least one bond linkage that comprises at leastone hetero atom of high electronegativity or S; and aryl groupsoptionally comprising at least one hetero atom selected from halogens,N, O, and S. Examples of moieties comprising at least one bond linkagethat comprises at least one hetero atom of high electronegativity or Sinclude, but are not limited to moieties comprising at least one amidebond, urethane bond, urea bond, thiourea bond, ether bond, or thioetherbond.

One of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ may be a branchingunit covalently linked to Q₃ through a branch B′ and a linker L, asshown in the formula (Formula 2) set forth directly below.

In Formula 2, T may be a substituted carbon, a substituted nitrogen, oran aryl optionally comprising at least one hetero atom selected from O,N and S. B′ has the formula (Formula 3) set forth directly below.

Formula 3—(CH₂)_(α′)-[A¹¹-(CH₂)_(β′)-]_(a′)[A¹²-(CH₂)_(γ)-]_(b′)[A¹³-(CH₂)_(δ′)-]_(c′)A¹⁴-

The —(CH₂)_(α′), of Formula 3 is covalently linked to T of Formula 2 andA¹⁴ of Formula 3 is covalently linked to L₃ of Formula 2. Independently,each of A¹¹, A¹², A¹³, and A¹⁴ may be a neutral or substantially neutralnucleic-acid-binding-enhancing-group (NABEG); a neutral branched alkyloptionally comprising at least one hetero atom selected from N, O and S;or at least one neutral saturated 5- or 6-membered ring optionallycomprising at least one hetero atom selected from N, O and S, asdescribed previously in connection with each of A¹, A², A³, A⁴, A⁵, A⁶,A⁷, A⁸, A⁹, and A¹⁰ of Formula 1. The subscripts associated with the(CH₂) methylene units, namely, α′, β′, γ′, and δ′, may be the same ordifferent, each independently indicating the size of the associatedmethylene unit and being zero or an integer from 1 to about 20. Thesubscripts associated with the bracketed portions of Formula 3, namely,a′, b′, and c′, may be the same or different, each independentlyindicating the size of the associated bracketed portion and being zeroor an integer from 1 to about 20.

In Formula 2, independently, L₃ may be a moiety comprising a singlebond; a polymethylene unit having 1 carbon to about 12 carbonsoptionally comprising at least one hetero atom selected from N, O and S;or an aryl group optionally comprising at least one hetero atom selectedfrom halogens, N, O and S, as described previously in connection witheach of the L components of Formula 1. The resulting molecule is atrimeric nucleic acid stain.

One of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ may be a branchingunit covalently linked to a reactive group R_(r) through a branch B′ anda linker L₃, as shown in the formula (Formula 4) set forth directlybelow.

In Formula 4, T, B′ and L₃ are defined as set forth above in connectionwith Formula 2 and Formula 3, with the exception that L₃ is covalentlybound to R_(r). The resulting molecule may be a nucleic acid stain asrepresented by Structure 3 above.

A dye with a reactive group -R_(r) can be used to label any of a widevariety of molecules that comprise a suitable functional group or arederivatized to comprise a suitable functional group. It is understoodthat the term “reactive group” can be used to refer to a “reactivegroup” or a “functional group” and that the term “functional group” canbe used to refer to a “reactive group” or a “functional group.” Eitherterm may refer, and both terms may refer, to a bond-forming group on adye, or to a bond-forming group on the substrate molecule to be labeled.Here, by way of convenience, but not limitation, a bond-forming group onthe dye will generally be referred to as a reactive group and abond-forming group on the substrate molecule will generally be referredto as a functional group. Merely by way of example, a dye with areactive group or functional group -R_(r) may have up to one positivecharge.

In general, conjugation of a dye to a substrate molecule may confer anucleic acid-detection property of the dye on the conjugated substratemolecule. The reactive group and the functional group are typically anelectrophile and a nucleophile, respectively, that can form a covalentbond. According to one alternative, the reactive group is aphotoactivatable group capable of reacting with a hydrocarbon moleculeupon ultraviolet photoactivation or photolysis. According to anotheralternative, the reactive group is a dienophile capable of reacting witha conjugated diene via a Diels-Alder reaction. According to yet anotheralternative, the reactive group is a 1,3-diene capable of reacting witha dienophile. Still other reactive group/functional group pairs may beselected based on Staudinger chemistry or the reaction between an azidogroup and a terminal alkyne (the so-called Click chemistry). Merely byway of example, examples of useful reactive groups, functional groups,and corresponding linkages are listed below in Table 1.

TABLE 1 Examples of Reactive Groups, Functional Groups, and CovalentLinkages Electrophilic Group Nucleophilic Group Resulting CovalentLinkage activated esters* amines/anilines Carboxamides acrylamidesThiols Thioethers acyl azides** amines/anilines Carboxamides acylhalides amines/anilines Carboxamides acyl halides Alcohols/phenolsEsters acyl nitriles Alcohols/phenols Esters acyl nitrilesamines/anilines Carboxamides aldehydes amines/anilines Imines aldehydesor ketones Hydrazines Hydrazones aldehydes or ketones HydroxylaminesOximes alkyl halides amines/anilines alkyl amines alkyl halidescarboxylic acids Esters alkyl halides Thiols Thioethers alkyl halidesalcohols/phenols Esters alkyl sulfonates Thiols Thioethers alkylsulfonates carboxylic acids Esters alkyl sulfonates alcohols/phenolsEsters anhydrides alcohols/phenols Esters anhydrides amines/anilinesCarboxamides aryl halides Thiols Thiophenols aryl halides Amines arylamines aziridines Thiols Thioethers boronates Glycols boronate esterscarboxylic acids amines/anilines Carboxamides carboxylic acids AlcoholsEsters carboxylic acids Hydrazines Hydrazides carbodiimides carboxylicacids N-acylureas or anhydrides diazoalkanes carboxylic acids Estersepoxides Thiols Thioethers haloacetamides Thiols Thioethershalotriazines amines/anilines Aminotrizaines halotriazinesalcohols/phenols triazinyl ethers imido esters amines/anilines Amidinesisocyanates amines/anilines Ureas isocyanates alcohols/phenols Urethanesisothiocyanates amines/anilines Thioureas maleimides Thiols Thioethersphosphoramidites Alcohols phosphite esters silyl halides Alcohols silylethers sulfonate esters amines/anilines alkyl amines sulfonate estersThiols Thioethers sulfonate esters carboxylic acids Esters sulfonateesters Alcohols Ethers sulfonyl halides amines/anilines Sulfonamidessulfonyl halides phenols/alcohols sulfonate esters *Activated esters, asunderstood in the art, generally have the formula —COΩ, where Ω is agood leaving group, such as succinimidyloxy (—OC₄H₄O₂),sulfosuccinimidyloxy (—OC₄H₃O₂—SO₃H), or -1-oxybenzotriazolyl(—OC₆H₄N₃), for example; or an aryloxy group or aryloxy substituted oneor more times by electron-withdrawing substituent(s), such as nitro,fluoro, chloro, cyano, trifluoromethyl, or combinations thereof, forexample, used to form activated aryl esters; or a carboxylic acidactivated by a carbodiimide to form an anhydride or mixed anhydride—OCOR^(a) or —OCNR^(a)NHR^(b) ,where R^(a) and R^(b), which may be thesame or different, are independently C₁-C₆ alkyl, C₁-C₆ perfluoroalkyl,or C₁-C₆ alkoxy; or cyclohexyl, 3-dimethylaminopropyl, orN-morpholinoethyl. **Acyl azides can also rearrange to isocyanates.

The reactive group may be one that will react with an amine, a thiol, oran aldehyde. The reactive group may be an amine-reactive group, such asa succinimidyl ester, for example, or a thiol-reactive group, such as amaleimide, a haloacetamide, or a methanethio-sulfonate (MTS), forexample, or an aldehyde-reactive group, such as an amine, an aminooxy,or a hydrazide, for example.

A reactive dye may be conjugated to any of a wide variety of substratemolecules. For example, a suitable substrate may be a nucleotide, anoligonucleotide, a peptide, a protein, a hapten, a drug, amicroparticle, a synthetic polymer, a natural polymer, a biologicalcell, a virus, a molecule of a solid surface, such as the surface of asilicon wafer, the surface of a polypropylene substrate or container, orthe like, for example. A molecule to be labeled may be a nucleotide, anoligonucleotide, a peptide, or a molecule that may interact with anucleic acid. For example, DNA-binding dyes have been used to label anoligonucleotide-based probe for qPCR applications. Shiguro, T., et al,Nucleic Acids Res. 24: 4992-7 (1996).

A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰, which may be the same ordifferent, may, independently, be NABEGs selected from moietiescomprising at least one bond linkage that comprises at least one heteroatom of high electronegativity or S; and aryl groups optionallycomprising at least one hetero atom selected from halogens, N, O, and S.Examples of moieties comprising at least one bond linkage that comprisesat least one hetero atom of high electronegativity or S include, but arenot limited to moieties comprising at least one amide bond, urethanebond, urea bond, thiourea bond, ether bond, or thioether bond. A¹, A²,A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ may be such that BRIDGE comprises atmost one positive charge, or is substantially neutral, and in the lattercase, each of these constituents may itself be substantially neutral,which includes actual neutrality.

BRIDGE may comprise any suitable number of non-hydrogen atoms, aspreviously described, such as from about 10 to about 100 non-hydrogenatoms, for example, or from about 12 to about 60 non-hydrogen atoms forthe dimeric dyes, and from about 20 to about 100 non-hydrogen atoms forthe trimeric dyes. For example, BRIDGE may have from about 15 to about40 non-hydrogen atoms for the dimeric dyes, and from about 30 to about70 non-hydrogen atoms for the trimeric dyes.

Merely by way of example, BRIDGE may have the formula (Formula 5) setforth directly below.

Formula 5—(CH₂)_(x)—C(═O)NH—(CH₂)_(α)—[O—(CH₂)_(β)]_(b)[O—(CH₂)_(γ)]_(c)—NH(O═C)—(CH₂)_(x)—In one such case, for example, L₁ of BRIDGE is —(CH₂)_(x)— and L₂ ofBRIDGE is —(CH₂)_(x)—, where each x, independently, is an integerselected from 1 to 11, inclusive; A¹ of BRIDGE is —C(═O)NH—; a of BRIDGEis 1; A² of BRIDGE is —O—; A³ of BRIDGE is —O—; α may be an integerselected from 2 to about 20, inclusive; each of β and γ, independently,may be 2 or 3; b may be zero or an integer selected from 2 to about 20;and c may be zero or 1; each of d, e, f, g, h and i of BRIDGE is 0; andA¹⁰ of BRIDGE is —NH(O═C)C—. Merely by way of example, BRIDGE may be asjust described, wherein c is 1. Further, merely by way of example,BRIDGE may be as just described, wherein c is 1, and further, wherein xmay be 5; α and γ may be the same and may be 2 or 3; β may be 2; and bmay be 0, 1, 2 or 3.Monomeric Dyes

Independently, each of the constituent monomeric dyes or functionalmolecules, Q1, Q2, and Q3, used for the dimeric and trimeric dyes may beselected from: 1) fluorescent nucleic acid dyes; 2) non-fluorescent,nucleic acid-binding molecules; 3) fluorescent, non-nucleic acid dyes;and 4) non-fluorescent, non-nucleic acid dyes. In general, Q1, Q2 and Q3may be selected and covalently linked via BRIDGE in a manner toencourage or to ensure intramolecular dimer formation in the absence ofDNA and formation of highly fluorescent DNA-dye complexes upon DNAbinding. Intramolecular dimer formation may be sufficient to provide theuseful hairpin conformation of a dimeric dye, as previously described.Such a dimeric dye may possess desirable properties, such as lowbackground fluorescence and low PCR inhibition, for example. Aspreviously described, it is possible to use a dimeric dye as describedherein at a relatively high concentration to generate a desirable, orstrong, fluorescent signal.

Intramolecular dimer formation may be confirmed by comparing theabsorption spectra of a dimeric dye or trimeric dye in an aqueoussolution with the absorption spectra of the related monomeric dye ordyes also in an aqueous solution. Any intramolecular dimer formation dyeshould cause the spectra of the component monomeric dyes in the dimer ortrimer to be shifted significantly relative to the spectra of therelated monomeric dye(s). In this regard, a significant shift may beabout 10 nm or more, by way of example. For example, in FIG. 2, thespectra associated with AOAO-7 are shifted significantly relative to thespectra of DMAO.

When the intramolecular dimer formation is a H-dimer formation, thespectra will usually undergo a significant blue shift. In this regard, asignificant shift may be about 10 nm or more, by way of example. Othertypes of intramolecular dimer formation are also possible and may resultin spectral shift in another direction, in spectral shifts in separatedirections for each of the component monomeric dyes, in an insignificantspectral shift, or in no spectral shift. In this regard, aninsignificant shift may be about 5 nm or less, by way of example. Ingeneral, when there is no significant spectral shift observed, otheranalytical techniques may be employed to confirm the formation of anyintramolecular dimer. Such analytical techniques include, but are notlimited to, nuclear magnetic resonance (NMR) spectroscopy, infraredspectroscopy, and fluorescence spectroscopy, for example. Anyintramolecular dye aggregation that results in a hairpin structure isgenerally desirable.

Various combinations of Q1, Q2, and Q3 may be useful or desirable.Merely by way of example, a dimeric dye may be constructed via fivedifferent combinations of Q1 and Q2, as schematically shown in FIG. 10.Further by way of example, examples of prepared dyes and associatedintermediates are listed below in Table 2.

TABLE 2 Prepared Fluorescent Nucleic Acid Dyes SPACER LENGTH No. NameStructure MWt. (ATOMS) 1 DMAO

478.41 N/A 2 TMAO

620.35 N/A 3 AO-3N

705.16 N/A 4 AO-2N

493.43 N/A 5 PMAO

691.47 N/A 6 AOAO-1

926.76 10 7 AOAO-2

1124.03 21 8 AOAO-3

1038.88 16 9 AOAO-2Q

1252.71 11 10 AOAO-4

1041.95 14 11 AOAO-5

896.73 8 12 AOAO-6

1010.92 16 13 AOAO-7

1080.96 19 14 TOTO-3

1088.94 16 15 AOAO-8

1064.92  16* 16 AOAO-9

1229 25 17 AOAO-10

1313.24 31 18 AOAO-11

1123 22 19 AOAO-12

1082.94 19 20 AOAO-13

1215.14 27 21 AOAO-14

1621.61 53 22 AOAO-12R

1132.95 19 23 AOTO-3

1094.99 16 24 TOTO-12

1146.23 20 25 TO(3)TO(3)-12

1245.34 20 26 TO(3)TO(3)-2

1302.44 22 27 AORO-7

1320.25 21 28 RORO-12

1550.51 22 29 TOTO-13

1248 27 30 STST-27

1116 27 31 STST-19

1000.8 19 32 AOAO-47

1547.6 47 33 AOAO-67

1864 67 33 AOAO-113

2541 113  35 ET-27

1239 27 36 STST-21N

1041 21

While many of the structures shown in Table 2 show one or more iodideanion(s), any other appropriate anion(s), such as those describedherein, such as chloride anion(s), merely by way of example, may be usedin place of the iodide anions shown.

A dimeric dye of the invention may comprise a fluorescent nucleic aciddye Q₁ and a fluorescent nucleic acid dye Q₂, wherein Q₁ and Q₂ may bethe same or different. When Q₁ and Q₂ are the same, the resulting dye isa homodimer, such as any of Dye Nos. 6-22, 24-26, and 28-36 of Table 2,merely by way of example. When Q₁ and Q₂ are different fluorescentnucleic acid dyes that have similar absorption and emission spectra, theresulting dimer is a heterodimer, such as that of Dye No. 23 of Table 2,merely by way of example. Such a heterodimer is functionally similar toa homodimer. In either case, both Q₁ and Q₂ are reporter dyes, such thatupon DNA binding, they both contribute to the detected fluorescentsignal, as schematically illustrated in Combination A of FIG. 10.Alternatively, a heterodimeric dye may comprise two differentfluorescent nucleic acid dyes that have substantially differentabsorption and emission spectra. In this case, only one of the two dyesis selected as a reporter dye.

Q₁ and Q₂ may form a fluorescence resonance energy transfer (FRET) pair.In this case, the dye with the shorter wavelength acts as a fluorescencedonor dye, while the dye with the longer wavelength acts as an acceptoror reporter dye. For efficient FRET to occur, the emission spectrum andthe absorption of the donor dye need to overlap sufficiently. Furtherdiscussions of FRET are provided in Förster, Ann. Phys. (1948) andStryer, et al., Proc. Natl. Acad. Sci. (1967). A FRET-based dye allowsfor excitation at one wavelength and re-emission of fluorescence at asubstantially longer wavelength.

When a heterodimer comprising Q₁ and Q₂ of substantially differentspectra is not a FRET-based dye, either one of Q₁ and Q₂, but not bothat the same time, may be selected as a reporter dye. The othernon-reporter dye serves as a partner for the necessary intramoleculardimer formation and provides additional nucleic acid binding ability forthe dimeric dye. An example of a heterodimeric dye having one reporterdye, a fluorescent nucleic acid dye Q₁, and one non-reporter dye, anon-fluorescent nucleic acid-binding molecule Q₂, is schematicallyillustrated in Combination B of FIG. 10.

A heterodimeric dye may comprise a non-fluorescent nucleic acid-bindingmolecule Q₁ and a fluorescent non-nucleic acid dye Q₂. Here, Q₂ is thereporter dye, while Q₁ serves as a DNA anchoring dye and a pairingpartner for the necessary intramolecular dimer formation. The DNAbinding mode for this type of heterodimer is schematically illustratedin Combination C of FIG. 10.

A heterodimeric dye may comprise a fluorescent nucleic acid dye Q₁ and anon-fluorescent non-nucleic acid dye Q₂. In such a case, Q₁ is thereporter dye and Q₂ serves as a partner for the necessary intramoleculardimer formation. The DNA binding mode for this type of heterodimer isschematically illustrated in Combination D of FIG. 10.

A heterodimeric dye may comprise a fluorescent nucleic acid dye Q₁ and afluorescent non-nucleic acid dye Q₂. If Q₁ and Q₂ have similarabsorption and emission spectra, both Q₁ and Q₂ are reporter dyes,although only Q₁ is bound to the nucleic acids. The DNA binding mode forthis type of heterodimer is schematically illustrated in Combination Eof FIG. 10. When Q₁ and Q₂ form a FRET pair, the dye with the shorterwavelength acts as the fluorescence donor dye, while the dye with thelonger wavelength acts as the acceptor or reporter dye. When Q₁ and Q₂are substantially different in spectra and are not a FRET pair, eitherone of Q₁ and Q₂, but not both at the same time, may be selected as areporter dye. An example of this latter case is the heterodimer AORO-7(Dye No. 27 of Table 2), which comprises AO with an absorption peak andan emission peak at 503 nm and 523 nm (DNA), respectively, and arosamine dye with an absorption peak and an emission peak and at 600 nmand ˜620 nm, respectively, as shown in FIGS. 12 and 13. FIG. 14 shows aPCR amplification plot using AORO-7, with the fluorescent non-nucleicrosamine dye component chosen as the reporter dye by using channel no. 3on an iCycler IQ Multiple-Color Real-Time PCR Detection System fromBio-Rad Laboratories (Hercules, Calif.).

A dimeric dye may comprise a pair of monomeric dyes selected from twoidentical fluorescent nucleic acid dyes and two different fluorescentnucleic acid dyes.

A trimeric dye may comprise a fluorescent nucleic acid dye Q₁, afluorescent nucleic acid dye Q₂, a fluorescent nucleic acid dye Q₃,wherein Q₁, Q₂ and Q₃ may be the same or different. For example, Q₁, Q₂,and Q₃ may be the same fluorescent nucleic acid dye. A trimeric dye maycomprise a fluorescent nucleic acid dye Q₁, a fluorescent nucleic aciddye Q₂, and a fluorescent non-nucleic acid dye Q₃, wherein Q₁ and Q₂serve as DNA anchoring molecules and Q₃ is a reporter dye. A trimericdye of the invention may comprise a non-fluorescent nucleic acid-bindingmolecule Q₁, a non-fluorescent nucleic acid-binding molecule Q₂, and athird fluorescent non-nucleic acid dye Q₃, wherein Q₁ and Q₂ serve asDNA anchoring molecules and Q₃ is a reporter dye.

Fluorescent nucleic acid dyes, non-fluorescent nucleic acid-bindingmolecules, fluorescent non-nucleic acid dyes, non-fluorescentnon-nucleic acid dyes, and examples thereof, are further describedbelow.

Fluorescent Nucleic Acid Dyes

Examples of a monomeric fluorescent nucleic acid dye suitable forconstructing dyes include, but are not limited to, an acridine dye, anasymmetric cyanine-based nucleic acid stain, a phenanthridinium dye, asymmetric cyanine nucleic stain, a derivative of DAPI, and a derivativeof a Hoechst dye. DAPI and Hoechst dyes generally cannot be directlyattached to BRIDGE because they do not possess a reactive group for bondformation. In this context, a derivative refers to a base dye, such asDAPI or a Hoechst dye, that is modified sufficiently for bond formation,such as by addition of a reactive group, by way of example.

Acridine Dyes

Merely by way of example, the monomeric fluorescent nucleic acid dye maybe an acridine dye having the general structure (Structure 4) set forthdirectly below.

Acridine orange (AO) is an acridine dye that stains dsDNA with greenfluorescence and stains RNA with red fluorescence. Traganos, et al., J.Histochem. Cytochem. 25(1), 46 (1977). Unlike some other acridine dyes,AO has a high extinction coefficient (>50,000) and a long absorptionwavelength (λ_(abs)=500 nm (DNA bound)). However, the affinity of AO fornucleic acid is very low and the dye has significant intrinsicfluorescence in the absence of nucleic acids. In this regard, the levelof intrinsic fluorescence may be significant in that it precludes thedye from being used in detecting nucleic acid at a low level, such as inthe low nanogram/mL range, for example, or in detecting nucleic acid ingels without a destaining step, for example. Consequently, AO itself isof little utility for DNA or RNA quantification, particularly for highlysensitive DNA detection associated with applications such as real-timeqPCR.

An acridine dye may comprise any of a variety of substituents at variouspositions on the ring structure. The nature of a substituent and itssubstitution position may strongly affect the spectral properties of thedye produced. In general, electron-donating substituents at the 3- and6-positions and an electron-withdrawing substituent at the 9-positiontypically red-shift the absorption and emission spectra of the dye.Examples of a typical electron-donating group include, but are notlimited to, an amino group, a hydroxyl group, an alkoxy group, and analkylmercapto group. Examples of a typical electron-withdrawing groupinclude, but are not limited to, a cyano group, a perfluoroalkyl group,a carboxamido group, a sulfonamide group, a nitro group, and a halogengroup. Any additional ring structure fused with the core structure willalso increase the wavelengths of the dye produced.

Various portions of Structure 4 are now described. In Structure 4, as invarious other monomeric dye structures provided or described herein, asymbol of “R” followed by a subscript, such as R₁, merely by way ofexample, may indicate a substituent of the structure that is not part ofBRIDGE, or may represent where BRIDGE attaches to the structure, inwhich case, it is not a substituent of the structure. Each R₁,independently, may be H; an alkyl or alkenyl having 1 carbon to 6carbons; a halogen; —OR₄; —SR_(S); —NR₆R₇; —CN; —NH(C═O)R₈; —NHS(═O)₂R₉;or —C(═O)NHR₁₀; any adjacent pair of R₁s optionally forms a 5- or6-membered saturated or unsaturated ring, which further optionallycomprises at least one hetero atom selected from N, O and S; and one ofthe R₁s is -L-R_(r), as previously described, or one of the R₁Srepresents where BRIDGE attaches to the structure, in which case, thatR₁ is merely representative and not actually a substituent of themonomeric dye. In any case where R₁ involves at least one of R₄, R₅, R₆,R₇, R₈, R₉, and R₁₀, any applicable one of same is independently H or analkyl having 1 carbon to 6 carbons, and for any applicable pair ofadjacent R₆ and R₇, independently, R₆ and R₇ may in combination form a5- or 6-membered saturated or unsaturated ring, which optionallycomprises at least one hetero atom selected from N and O.

Typically, R₂ is H; an alkyl or alkenyl having 1 carbon to 6 carbons; anaryl optionally comprising at least one hetero atom selected fromhalogens, N, O and S; a halogen; —OR₁₁; —SR₁₂; —NHR₁₃; —CN; or—C(═O)NHR₁₄; or represents where BRIDGE attaches to the structure. Inany case where R₂ involves at least one of R₁₁, R₁₂, R₁₃ and R₁₄, anyapplicable one of same is independently H or alkyl having 1 carbon to 6carbons.

Typically, R₃ is H; or an alkyl having 1 carbon to 6 carbons; orrepresents where BRIDGE attaches to the structure.

Ψ is an anion, such as an anion that balances positive charge(s)associated with the dye, for example. Ψ may be biologically compatible.Examples of a suitable anion include, but are not limited to, a halide,a sulfate, a phosphate, a perchlorate, a tetrafluoroborate, and ahexafluorophosphate. Merely by way of example, the anion may be chlorideor iodide.

Only one of R₁, R₂ and R₃ must represent where BRIDGE attaches to thestructure. Merely by way of example, one of R₂ and R₃ may representwhere BRIDGE attaches to the structure. As described herein, BRIDGE maybe covalently linked to a monomeric acridine dye, such as any such dyedescribed herein, and to another suitable monomeric dye, to form adimeric dye, or to two other suitable monomeric dyes to form a trimericdye. Generally, only one of R₁, R₂ and R₃ may be optionally-L-R_(r), aspreviously described. A dimeric dye or a trimeric dye may comprise onlyone-L-R_(r).

Merely by way of example, the monomeric acridine dye may have thestructure (Structure 5) set forth directly below.

In Structure 5, generally, each R₁, independently, is H, or a C1-C2alkyl; one of R₂ and R₃ represents where BRIDGE attaches to thestructure; optionally, one of R₂ and R₃ is -L-R_(r), as previouslydescribed; when R₂ does not represent where BRIDGE attaches to thestructure and is not L-R_(r), R₂ is selected from H, —CH₃, —NH₂, —NHCH₃,—CN, and —C(═O)NH₂; when R₃ does not represent where BRIDGE attaches tothe structure and is not L-R_(r), R₃ is selected from H or —CH₃; each ofR₆ and R₇, independently, is H, or a C1-C2 alkyl; and Ψ is an anion, aspreviously described. Merely by way of example, for each pair ofadjacent R₆ or R₇ and R₁, independently, R₆ or R₇ and R₁ may incombination form a 5- or 6-membered, saturated or unsaturated ring.

In one example, the monomeric acridine dye, as represented by Structure5, may be such that each R₁ is H; R₂ is H; R₃ represents where BRIDGEattaches to the structure; each R₆ is —CH₃; each R₇ is —CH₃; and Ψ is ananion, as previously described.

Merely by way of example, a dimeric dye may comprise two identicalmonomeric acridine dye molecules of Structure 5 and BRIDGE of Formula 5.

Asymmetric Cyanine Dyes

Merely by way of example, the monomeric fluorescent nucleic acid dye maybe an asymmetric cyanine dye having the general structure (Structure 6)set forth directly below.

The general structure (Structure 6, above) of asymmetric cyanine dyescomprises a heterocyclic ring that is a substituted benzazolium ring; amethane or polymethine bridge; and a heterocyclic ring that is asubstituted pyridinium or quinolinium ring. The dotted line in thestructure represents the atoms necessary to form one or more fusedaromatic ring(s), optionally incorporating one or more nitrogen(s),which may or may not be quaternized. When the dotted line represents a6-membered ring comprising one or more nitrogen atom(s), the resultingfused ring is called an aza-benzole ring.

In Structure 6, in general, each of R₁ and R₁′ on the benzazolium ring,independently, is H; alkyl or alkenyl having 1 carbon to 6 carbons; ahalogen; —OR₉; —SR₁₀; —NR₁₁R₁₂; —CN; —NH(C═O)R₁₃; —NHS(═O)₂R₁₄; or—C(═O)NHR₁₅. Merely by way of example, one of R₁ and R₁′ may be asubstituent that is meta to X or to the benzazole nitrogen, wherein thesubstituent confers at least one desirable property as further describedbelow. In any case where R₁ or R₁′ involves at least one of R₉, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅, any applicable one of same, independently,is H; or alkyl having 1 carbon to 12 carbons, optionally incorporating 1to 2 nitrogen(s); or an aryl; and any applicable R₁₁ and R₁₂ may incombination form a 5- or 6-membered saturated or unsaturated ring, whichoptionally comprises at least one hetero atom selected from N and O.

As mentioned above, one of R₁ and R₁′ of Structure 6 may be asubstituent that confers at least one desirable property to the dye. Onesuch desirable property is DNA minor groove-binding. A minorgroove-binding molecule typically has a structure with a crescent shapethat fits into the minor groove of a double-stranded DNA. Examples of aDNA minor groove-binding dye molecule or non-dye molecule, which mayinclude a natural molecule, include, but are not limited to, DAPI, aHoechst dye, distamycin A, netropsin, and any of numerous syntheticminor groove-binders based on polyamides of N-methylpyrrole andN-methylimidazole. Catalog of Biotium, Inc. (Hayward, Calif. (CA)),2005-2006; Boger, et al., Acc. Chem. Res. 37, 61 (2004); and Dervan, P.B., Bioorg. & Med. Chem. 9, 2215 (2001). The crescent shape of a minorgroove-binder is typically created by meta-substitution of a 5- or6-membered ring with a minor groove-binder substituent, which includes,but is not limited to, a substituted or an unsubstitutedbenzoxazol-2-yl, a substituted or an unsubstituted benzimidazol-2-yl, asubstituted or an unsubstituted benzothiazol-2-yl, a substituted or anunsubstituted imidazol-2-yl, a substituted or an unsubstitutedoxazol-2-yl, a substituted or an unsubstituted thiazol-2-yl, asubstituted or an unsubstituted N-methylpyrrolyl-2-aminocarbonyl, asubstituted or an unsubstituted N-methylpyrrolyl-3-carboxamido, asubstituted or an unsubstituted 1-methylimidazol-2-carboxamido, asubstituted or an unsubstituted 1-methylimidazol-4-aminocarbonyl, asubstituted or an unsubstituted phenyl, a substituted or anunsubstituted pyridyl, a substituted or an unsubstituted pyrazinyl, anda substituted or an unsubstituted triazinyl. A DNA dye may bemeta-substituted by a minor groove-binder substituent as described inU.S. Patent Application Publication No. 2004/0132046.

One of R₁ and R₁′ may be -L-R_(r), as previously described. One of R₁and R₁′ may represent where BRIDGE attaches to the structure.

X is selected from O and S. In general, a dye wherein X is S has longerabsorption and emission wavelengths than a similar dye wherein X is O.

R₂ may be methyl or ethyl, or may represent wherein BRIDGE attaches tothe structure. Merely by way of example, R₂ may be methyl or ethyl.

The subscript n represents a number of double bond units in any methinebridge and is selected from 0, 1, and 2. Typically, a dye with a longermethine bridge will have longer wavelengths than a dye with a shortermethine bridge. Merely by way of example, n may be 0 or 1.

Substitutents R₃, R₄, and R₅ are independently H or —CH₃. Optionally,any adjacent pair of these substitutents may form a 5- or 6-memberedring. Merely by way of example, R₃, R₄, and R₅ may be H.

In general, independently, each of substituents R₆, R₈, and R₈′ may beH; an alkyl or alkenyl having 1 carbon to 10 carbons, optionallycomprising at least one hetero atom selected from N, O, and S; ahalogen; —OR₁₆; —SR₁₆; —NR₁₆R₁₇; a substituted or unsubstituted aryl,optionally comprising 1 to 3 hetero atom(s) selected from halogens, N,O, and S. R₈ and R₈′ may in combination form a fused aromatic ring,which may be further substituted 1 to 4 time(s), independently, by C1-C2alkyl, C1-C2 alkoxy, C1-C2 alkylmercapto, or a halogen. In any case inwhich any of R₆, R₈, and R₈′ involve at least one of R₁₆ and R₁₇, anyapplicable one of same, independently, is H; or alkyl having 1 carbon to12 carbons, optionally incorporating 1 to 2 nitrogen(s); or an aryl; andany applicable R₁₆ and R₁₇ may in combination form a 5- or 6-memberedsaturated or unsaturated ring, which optionally comprises at least onehetero atom selected from N and O.

R₆ may represent where BRIDGE attaches to the structure. R₆ may be a-L-R_(r), as previously described.

R₇ is selected from H; an alkyl or alkenyl having 1 carbon to 10carbons, optionally comprising an aryl and at least one hetero atomselected from N, O, and S; a substituted or unsubstituted aryloptionally comprising 1 to 3 hetero atom(s) selected from halogens, N,O, and S; a -L-R_(r), as previously described; or may represent whereBRIDGE attaches to the structure.

Ψ is an anion, as previously described herein.

Only one of R₁, R₁′, R₆, R₇ and R₈ must represent where BRIDGE attachesto the structure. As described herein, BRIDGE may covalently link themonomeric asymmetric cyanine dye and another suitable monomeric dye toform a dimeric dye, or the monomeric asymmetric cyanine dye and twoother suitable monomeric dyes to form a trimeric dye. Generally, onlyone of R₁, R₁′, R₆, R₇ and R₈ may optionally be -L-R_(r), as previouslydescribed. More typically, a dimeric dye or a trimeric dye may compriseonly one -L-R_(r).

Merely by way of example, an asymmetric cyanine dye may have thestructure (Structure 7) set forth directly below, wherein each of R₁′,R₆, R₇, R₈ and R₈′ is as previously described in connection withStructure 6.

By way of example, the asymmetric cyanine dye, as represented byStructure 7, may be such that R₁′ is H; alkyl or alkenyl having 1 carbonto 6 carbons; a halogen; —OR₉; —SR₁₀; —NR₁₁R₁₂; —CN; —NH(C═O)R₁₃;—NHS(═O)₂R₁₄; —C(═O)NHR₁₅; a substituent associated with minor groovebinding; or -L-R_(r), as previously described; or represents whereBRIDGE attaches to the structure. Further, when R₁′ comprises at leastone of R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅, any said one of R₉, R₁₀,R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅, independently, is H or alkyl having 1 carbonto 12 carbons, optionally incorporating 1 to 2 nitrogen(s), or an aryl;and when R₁′ comprises R₁₁ and R₁₂, R₁₁ and R₁₂ may in combination forma 5- or 6-membered, saturated or unsaturated ring, which optionallycomprises at least one hetero atom selected from N and O. X may beselected from O and S and n may be selected from 0, 1, and 2. R₆ may beH; alkyl or alkenyl having 1 carbon to 10 carbons, optionally comprisingat least one hetero atom selected from N, O, and S; a halogen; —OR₁₆;—SR₁₆; —NR₁₆R₁₇; a substituted or an unsubstituted aryl, optionallycomprising 1 to 3 hetero atom(s) selected from halogens, N, O, and S; or-L-R_(r), as previously described; or may represent where BRIDGEattaches to the structure. R₇ may be H; alkyl or alkenyl having 1 carbonto 10 carbons, optionally comprising an aryl and at least one heteroatom selected from N, O, and S; a substituted or an unsubstituted aryloptionally comprising 1 to 3 hetero atom(s) selected from halogens, N,O, and S; or -L-R_(r), as previously described; or may represent whereBRIDGE attaches to the structure. R₈′ may be H; alkyl or alkenyl having1 carbon to 10 carbons, optionally comprising at least one hetero atomselected from N, O, and S; a halogen; —OR₁₆; —SR₁₆; —NR₁₆R₁₇; or asubstituted or an unsubstituted aryl, optionally comprising 1 to 3hetero atom(s) selected from halogens, N, O, and S; or -L-R_(r), aspreviously described; or may represent where BRIDGE attaches to thestructure. R₈′ may be H; alkyl or alkenyl having 1 carbon to 10 carbons,inclusive, optionally comprising at least one hetero atom selected fromN, O, and S; a halogen; —OR₁₆; —SR₁₆; —NR₁₆R₁₇; or a substituted or anunsubstituted aryl, optionally comprising 1 to 3 hetero atom(s) selectedfrom halogens, N, O, and S. R₈ and R₈′ may in combination form a fusedaromatic ring, which may be further substituted 1 to 4 time(s),independently, by C1-C2 alkyl, C1-C2 alkoxy, C1-C2 alkylmercapto, or ahalogen. For any R₆, R₈, or R₈′ that comprises at least one of R₁₆ andR₁₇, any said one of R₁₆ and R₁₇ thereof, independently, may be H; alkylhaving 1 carbon to 12 carbons, optionally incorporating 1 to 2nitrogen(s) or an aryl. For any R₆, R₈, and R₈′ that comprises R₁₆ andR₁₇, R₁₆ and R₁₇ thereof may in combination form a 5- or 6-memberedsaturated or unsaturated ring, which optionally comprises at least onehetero atom selected from N and O. Only one of R₁′, R₆, R₇ and R₈represents where BRIDGE attaches to the structure. Generally, only oneof R₁′, R₆, R₇ and R₈ may optionally be -L-R_(r), as previouslydescribed. Ψ is an anion, as previously described.

In one example, an asymmetric cyanine dye has the structure (Structure8) set forth directly below, wherein R₇ represents where BRIDGE attachesto the structure and Ψ is an anion, as previously described.

Merely by way of example, a dimeric dye may comprise two identicalmonomeric asymmetric cyanine dye molecules of Structure 8 and BRIDGE ofFormula 5.

Merely by way of example, in a fluorescent nucleic acid dye, such asthat of Structure 1, for example, when the Q1 dye constituent is anasymmetric cyanine dye, such as any of Structures 6-8, for example, andthe Q2 dye constituent is an asymmetric cyanine dye, such as any ofStructures 6-8, for example, a sum of a, b, c, d, e, f, g, h, and i ofBRIDGE, such as BRIDGE of Formula 1, for example, may be greater thanthree, or at least one of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰ ofBRIDGE, such as BRIDGE of Formula 1, for example, may be a NABEGcomprising a moiety that comprises at least one bond linkage thatcomprises at least one amide bond, urethane bond, urea bond, or thioureabond; or an aryl optionally comprising at least one hetero atom selectedfrom halogens, N, O, and S.

Phenanthridinium Dyes

Merely by way of example, the monomeric fluorescent nucleic acid dye maybe a phenanthridinium derivative, having the general structure(Structure 9) set forth directly below.

In general, R₁ may represent where BRIDGE attaches to the structure,although it will be understood that many variations of Structure 9 aboveare possible and contemplated herein, via a variety of techniques, suchas synthesis techniques that may provide for the attachment of BRIDGE tothe structure elsewhere or that may modify the structure to provide adye with any of various desirable wavelengths. Ψ is an anion, aspreviously described.

Merely by way of example, two monomeric phenanthridinium dye moleculesof Structure 9 in combination with BRIDGE of Formula 5 may form adimeric dye.

Merely by way of example, the monomeric fluorescent nucleic acid dye maybe a xanthene derivative, having the general structure (Structure 10)set forth directly below.

Certain cationically charged xanthene dyes are known to bind to nucleicacids. For example, pyronin Y, in which R₁, R₂, R₁′, and R₂′ are methyland R₃, R₃′, R₄, R₄′, and A are H, is a known fluorescent DNA bindingdye that has been used for DNA gel staining. Adkin, S., and Burmeister,M., Anal. Biochem. 240(1), 17 (1996). A dye having the general skeletonshown in Structure 10 above is expected to have similar nucleic acidstaining properties and to provide other fluorescent colors. Forexample, pyronin Y has an absorption maximum at 548 nm and an emissionmaximum at 565 nm, providing a red fluorescent color.

Merely by way of example, in general, each of R₁, R₂, R₁′, and R₂′,independently, may be H, or C1-C6, inclusive, alkyl, optionallyincorporating 1 to 2 hetero atom(s) selected from N and O. Furthermerely by way of example, independently, at least one of the pair R₁ andR₂ and the pair R₁′ and R₂′ may in combination form a 5- or 6-memberedring, optionally comprising one hetero atom selected from N and O. R₁and R₁′ may be the same and R₂ and R₂′ may be the same.

One of R₁, R₂, R₁′, and R₂′ may represent where BRIDGE attaches to thestructure. Optionally, one of R₁, R₂, R₁′, and R₂′ is -L-R_(r), aspreviously described.

Merely by way of example, R₃, R₃′, R₄, and R₄, independently, may be Hor C1-C3, inclusive, alkyl. R₃, R₃′, R₄, and R₄′ may be the same.Independently, at least one of the pair R₃ and R₁, the pair R₂ and R₄,the pair R₃′ and R₁′, and the pair R₄ and R₂′ may in combination form a5- or 6-membered ring, which may be saturated or unsaturated,substituted or unsubstituted.

A is a C1-C3 alkyl; or -L-R_(r), where L is a C1-C12 aliphatic linkerand R_(r) is a reactive group, as previously described; or representswhere BRIDGE attaches to the structure.

Only one of R₁, R₂, R₁′, R₂′ and A may represent where BRIDGE attachesto the structure.

Ψ is an anion, as previously described.

Two monomeric xanthene dye molecules of Structure 10 in combination withBRIDGE of Formula 5 may form a dimeric dye.

Other monomeric fluorescent nuclei acid stains, such as DAPI, DIPI, aHoechst dye, LDS 751, hydroxystilbamidine, a styryl dye, a merocyaninedye, a cyanine dye, or FluoroGold, merely by way of example, may besuitable for use or may be derivatized to be suitable for use asdescribed herein. Haugland, R. P., Handbook of Fluorescent Probes andResearch Products, 9^(th) edition. It will be understood that a largenumber of other monomeric nucleic acid dyes may be suitable for use ormay be derivatized to be suitable for use as described herein. The dyesmay either be directly conjugated to BRIDGE or be derivatized so thatthey can be conjugated to BRIDGE using synthesis knowledge.

Non-Fluorescent Nucleid Acid Dyes

In general, non-fluorescent nucleic acid-binding molecules are nucleicacid-binding molecules that are non-fluorescent or are too weaklyfluorescent to be useful as fluorescent nucleic acid dyes.Non-fluorescent nucleic acid-binding molecules include non-fluorescentnucleic acid-binding dyes and colorless synthetic or natural nucleicacid-binding molecules.

A number of non-fluorescent dyes have been used as colorimetric nucleicacid gel stains. Relative to the fluorescent nucleic acid stain ethidiumbromide, these non-fluorescent dyes usually have much lower detectionsensitivity, but are considered to be safer to use, such as safer interms of toxicity for use by humans, for example. Examples ofnon-fluorescent nucleic acid-binding dyes include, but are not limitedto, Nile Blue, Crystal Violet, Methylene Blue, Thionin, Methyl Green,Basic Blue 66, Basic Red 29, Indoline Blue, Safranin 0, Janus Green B,Pinacyanol, and Stains-All. Adkins, et al., Anal. Biochem. 240, 17(1996). Most of these dyes are available from Aldrich Chemical Company,Inc. (Milwaukee, Wis.).

Fluorescent Non-nucleic Acid Dyes

One of the monomeric dyes Q₁, Q₂, and Q₃ may be a fluorescentnon-nucleic acid dye. In general, all fluorescent dyes that are notnormally considered fluorescent nucleic acid dyes are consideredfluorescent non-nucleic acid dyes. Herein, the term “fluorescentnon-nucleic acid dye” generally refers to a fluorescent dye that is notnormally considered a nucleic acid dye. By way of example, the dye maynot normally be considered a fluorescent minor groove-binder or afluorescent intercalator. Further by way of example, while somefluorescent non-nucleic acid dyes may exhibit some weak interactionswith nucleic acids, these interactions are generally not sufficient tocause significant fluorescence spectral changes to make the dyes usefulfor nucleic acid detection.

Various fluorescent non-nucleic acid dyes are commercially availablefrom various sources, such as Biotium, Inc. (Hayward, Calif.). Examplesof a fluorescent non-nucleic acid dye include, but are not limited to, afluorescein dye, a sulfonated fluorescein dye, a rhodamine dye, asulfonated rhodamine dye, a cyanine dye, a sulfonated cyanine dye, acoumarine dye, a pyrene dye, an oxazine dye, and a Bodipy dye (MolecularProbes, Inc. (Eugene, Oreg.)). A suitable fluorescent non-nucleic aciddye may comprise a reactive group R_(r), as previously described. Asuitable fluorescent non-nucleic acid dyes may be derived such that itcomprises a reactive group R_(r). A suitable reactive dye is covalentlyattached to BRIDGE via R_(r) and a suitable functional group fromBRIDGE.

Selection of a suitable fluorescent non-nucleic acid dye may depend onthe other pairing monomeric dye or dyes. In general, a suitablefluorescent non-nucleic acid dye should be able to form anintramolecular dimer with the pairing dye or dyes. Intramolecular dimerformation is typically confirmed by a significant change in theabsorption spectrum of at least one of the component monomeric dyes inan aqueous media before and after the monomeric dye is covalently linkedto the other pairing monomeric dye or dyes by BRIDGE.

Non-Fluorescent Non-nucleic Acid Dyes

In general, the term “non-fluorescent non-nucleic acid dye” refers to adye that is neither fluorescent nor nucleic acid-binding. Such a dye isgenerally used as a fluorescence quencher in a FRET-based application.By way of example, a fluorogenic peptidase substrate has beenconstructed by covalently attaching a fluorescent donor dye to one endof a peptide, and a non-fluorescent non-nucleic acid dye, the quencher,to the other end of the peptide to quench the fluorescence of the donor.Upon enzymatic cleavage of the peptide, the donor and the quencher areseparated, thereby releasing the fluorescence signal. Further by way ofexample, a non-fluorescent non-nucleic acid dye has been used to designso-called TaqMan probes for qPCR. A TaqMan probe consists of anoligonucleotide, which is complimentary to a target DNA sequence, afluorescence donor dye attached to one end of the oligonucleotide, and aquencher attached to the other end of the oligonucleotide to quench thefluorescence of the donor dye. During real-time PCR, the labeledoligonucleotide binds to the target DNA, which causes theoligonucleotide to be enzymatically cleaved, thereby generating afluorescent signal.

A non-fluorescent non-nucleic acid dye may be used mainly as a pairingpartner for intramolecular dimer formation, which is responsible for therelease-on-demand DNA-binding mechanism. The non-fluorescent non-nucleicacid dye should be so chosen to ensure minimal FRET between it and thefluorescent nucleic acid-binding dye it pairs with following DNAbinding. In general, fluorescence loss of the fluorescent nucleic aciddye due to FRET should be minimal, such as no more than about 70% of thetotal emitted fluorescence, for example. The selection of thenon-fluorescent non-nucleic acid dye may be based on an evaluation ofthe emission spectrum of the fluorescent nucleic acid-binding dye andthe absorption spectrum of the non-fluorescent dye. Ideally, thesespectra should have minimal overlap so that the fluorescence signal lossdue to FRET is minimal, as described above. Another possible way tominimize FRET is to use sufficiently long BRIDGE so that thefluorescence signal loss due to FRET is minimal, as described above, asthe efficiency of FRET is dependent on the inverse sixth power of theintermolecular separation between the constituent dyes.

Examples of commercially available non-fluorescent quenchers which maybe useful include, but are not limited to, DABCYL from Fluka (Buchs,Switzerland), Black Hole Quencher (BHQ) from Biosearch Technologies,Inc. (Novato, Calif.), Eclipse Dark Quencher (DQ) from Epoch Biosciences(Bothell, Wash.), IOWA Black (IWB) from Integrated DNA Technologies(Skokie, Ill.), and QSY from Molecular Probes, Inc. (Eugene, Oreg.).

Method of Use

A nucleic acid dye described herein may be particularly useful inquantitative real-time PCR (qPCR). Using PCR coupled withfluorescence-based DNA detection via a fluorescent nucleic acid dye, onemay determine the amount of a product of a PCR process without having tostop a PCR run or to sample the reaction during a PCR run. Using qPCR,one may not only quantify the original amount of a DNA sample, but mayalso obtain sequence information. The sensitivity and specificity ofqPCR makes it highly useful in a number of practical applicationsincluding the diagnosis and prognosis of diseases, and theidentification of species in agriculture and forensic science.

The use of a dye in qPCR may involve adding a solution of the dye andother components suitable for a PCR reaction (such as an amplificationenzyme or enzymes, a primer or primers sufficient for amplification ofthe target nucleic acid sequence, and deoxynucleoside triphosphates, forexample) to a solution comprising a DNA sample in a tube, placing thesealed tube in a qPCR instrument, and recording the detected fluorescentsignal. The Ct value, or the number of cycles required for thefluorescence signal to reach an arbitrarily determined threshold value,may be recorded. The Ct value is linearly related to the log of the DNAsample copy number. Using a standard plot of Ct value and the log of DNAcopy number, one can determine the DNA copy number of a DNA sample basedon the Ct value. Merely by way of example, PCR amplification plots usingselected dyes are shown in FIGS. 8, 9, 11, and 14.

Other uses of a fluorescent nucleic acid dye include, but are notlimited to, DNA quantitation in solutions or gels, staining of nucleicacids in live or dead cells, and nucleic acid detection in microarrays.Generally, the use of a fluorescent nucleic acid dye may comprisecontacting the dye, optionally in combination with any additionalreagent(s), with a sample that comprises or is thought to comprise anucleic acid polymer; incubating the resulting mixture of dye and samplefor a sufficient amount of time to allow formation of dye-nucleic acidcomplexes; and detecting the fluorescent signal of the dye-nucleic acidcomplexes.

The dye may be prepared for suitable use as described herein. Merely byway of example, the dye may be made into a stock solution using anaqueous solvent or a water-miscible and biologically compatible organicsolvent at a concentration of greater than about 100 times that used inthe final staining solution. Examples of suitable aqueous solvents thatmay be used alone or in combination with a suitable organic solvent inthe making of a dye stock solution, include, but are not limited to,water, PBS buffer, and Tris buffer. Examples of suitable organicsolvents for the making of a dye stock solution, include, but are notlimited to, DMSO, DMF, methanol or ethanol. The stock solution is thendiluted into a staining solution with a desired final dye concentrationusing a suitable aqueous solvent, such as water or a biological buffer,for example. In general, the specific dye concentration for the stainingsolution may be determined by the nature of the sample to be analyzedand the nature of the analysis being performed. By way of example, ingeneral, a staining solution for use in connection with a cellularsample may have a dye concentration of about 1 nM or more, or up toabout 100 μM. Further by way of example, in general, a staining solutionfor use in connection with an electrophoretic gel may have a dyeconcentration of about 1 μM or more, or up to about 50 μM.

A method of staining nucleic acids using a dye may be determined by thenature of the analysis being carried out. In the staining of nucleicacids in cellular or tissue samples, which may or may not be pre-fixed,the samples are usually incubated in a staining solution for a fewminutes to 2 hours to allow the dye to permeate the cell membranes andcombine with the nucleic acids. In some cases, nucleic acids may bepresent in the form of a solution comprising purified nucleic acids orcrude cell extracts. In such cases, in general, addition of a dye stocksolution to a nucleic acid solution should result in an instantaneouslydetectable fluorescence signal, the strength of which is proportional tothe amount of nucleic acid. By way of example, a DNA titration curve isshown in FIG. 7. The substantially linear relationship between theamount of DNA and fluorescence intensity can be used for quantitation ofDNA, or when cell extract is used, estimation of the number of cells. Incertain instances, a nucleic acid may be embedded in an inert matrix,such as a blot or gel, a test strip, for example, or attached to a solidsurface, such as a microarray chip or any other solid surface, forexample. In such cases, in general, staining is carried out by applyinga staining solution to the surface of the nucleic acid-comprisingmatrix, or to the surface of a microarray chip or other solid surface,and incubating for a period sufficient to allow formation of dye-nucleicacid complexes.

A fluorescent nucleic acid-dye complex may be detected either via itsemission or excitation. By way of example, the fluorescent nucleicacid-dye complex may be, and typically is, excited by a light withwavelength at or near the absorption maximum wavelength of the complex.Further by way of example, the nucleic acid-dye complex may be excitedby UV light with wavelength from 300 nm to 400 nm, which is a commonsource of excitation light available on most of the transluminators usedfor gel visualizing applications. By way of example, the fluorescentsignal may be detected via various instruments, such as plate readers,microscopes, fluorometers, quantum counters, and flow cytometers, forexample. Further by way of example, the fluorescent signal may be madeby visual methods, such as visual inspection or photographic recording,for example.

Synthesis

The synthesis of a dye may be described in terms of synthesis of themonomeric dye constituents, synthesis of BRIDGE, and conjugation of themonomeric dye constituents to BRIDGE. Syntheses of monomeric dyes andmonomeric dyes comprising a functional group or a reactive group are nowdescribed.

Synthesis of the Monomeric Dyes and Functional Molecules

Suitable monomeric dyes and monomeric dyes comprising a functional groupor a reactive group may be prepared from scratch by a known procedure orany suitable procedure, or by modifying commercially available materialthat already has a suitable or desirable core structure. Many monomericacridine dyes may be prepared from commercially available acridine dyes.A few examples of a commercially available acridine dye that may serveas suitable starting material for synthesis are set forth directlybelow.

Other acridine core structures may be prepared according to knownprocedures or any suitable procedures. Albert, A., The acridines: theirpreparation, physical, chemical, and biological properties and uses,Edward Arnold Ltd., London; Eldho, et al., Synth. Commun. 29, 4007(1999); and Joseph, et al., Bioconjugate Chem. 15, 1230 (2004). Anacridine core structure may be formed by condensing a suitablediphenylamine with a suitable carboxylic acid or a carboxylic acidequivalent in the presence of a Lewis acid, as schematically illustratedin Reaction 1 directly below.

In Reaction 1, R′, R″ and R′″ are suitable substituents, as furtherdescribed below. The diphenylamine starting material is eithercommercially available or may be synthesized from a suitable arylhalideand a suitable arylamine using a known method or any suitable method.Yang, et al., J. Organomet. Chem. 576, 125 (1999); Hartwig, et al., J.Org. Chem. 64, 5575 (1999); and Wolfe, et al., J. Org. Chem. 65, 1158(2000).

The nature of the substituents and the position where the substituentsare attached may have a profound effect on the spectral property of thedye. In general, electron-donating groups at the 2-, 3-, 6- and7-positions will increase the absorption and emission wavelengths of thedye. A typical electron-donating group may be an amino group, analkylamino group, a dialkylamino group, a hydroxyl group, an alkoxygroup, a thiol group, or an alkylthio group, by way of example. A moretypical electron-donating group may be an amino group, an alkylaminogroup, a dialkylamino group, or an alkoxy group, by way of example. Ingeneral, an electron-withdrawing group at the 9-position will increasethe absorption and emission wavelengths of the dye. A typicalelectron-withdrawing group may be a cyano group, a perfluoroalkyl group,an aminocarbonyl group, an alkylaminocarbonyl group, an alkylcarbonylgroup, an aldehyde group, an alkoxycarbonyl group, an aminosulfonatogroup, an alkylaminosulfonato group, or a halide group, by way ofexample. A more typical electron-withdrawing group may be a cyano group,a perfluoroalkyl group, or a halide group.

In general, once the acridine core structure is built, the 10-nitrogenis alkylated with a haloalkyl group, which typically comprises anadditional reactive group or a functional group that can be converted toa reactive group. The additional reactive group serves to conjugate theacridine dye to BRIDGE. Several ways of making monomeric acridine orangedyes with a suitable reactive group are schematically illustrated inScheme 1 directly below.

The 9-position of 10-alkylated acridine may be readily substituted witha cyano group, which can be further hydrolyzed to a carboxamide group,as schematically illustrated in Scheme 2 directly below.

Methods of preparing reactive monomeric asymmetric cyanine dyes havebeen described. Carreon, et al., Org. Lett. 6(4), 517 (2004). Such a dyemay have the structure (Structure 11) set forth directly below.

U.S. Pat. No. 5,863,753 discloses the preparation of a series ofreactive asymmetric cyanine dyes, including ones that have a substituentortho to the quinolinium or pyridinium nitrogen. Such a substituent,especially a cyclic substituent, ortho to the quinolinium or pyridiniumnitrogen, is said to confer desired properties to the asymmetric cyaninedyes, according to U.S. Pat. No. 5,436,134. These cyclically substitutedasymmetric cyanine dyes are commonly referred to as SYBR dyes. Zipper,et al., Nucleic Acids Res. 32 (12), e103 (2004). Some of the reactiveSYBR dyes are commercially available from Molecular Probes, Inc.(Eugene, Oreg.), although the exact structures of these dyes are notknown. Haugland, R. P., Handbook of Fluorescent Probes and ResearchChemicals, 9^(th) edition.

U.S. Patent Application Publication No. 2004/0132046 discloses methodsfor preparing monomeric asymmetric cyanine dyes with minorgroove-binding capability. In general, these dyes possess acrescent-shaped structure by virtue of having an additional benzazolylsubstitutent on the benzazolyl ring of the dyes. Similar monomeric dyeshaving a suitable reactive group may be prepared using similar methods,for example, as schematically illustrated in Scheme 3 directly below.

Reactive phenanthridinium dyes may be prepared from the commerciallyavailable 3,8-diamino-6-phenylphenanthridine, as schematicallyillustrated in Scheme 4 directly below.

Merely by way of example, Dye No. 35 of Table 2 may be prepared usingthe phenanthridinium intermediate with a reactive group shown in Scheme4 above.

Preparations of pyronin derivatives with a reactive group at the9-position may be carried out by condensing two equivalents ofm-aminophenol derivative with one equivalent of dicarboxylic anhydride,as schematically illustrated in Scheme 5 directly below.

Many monomeric non-fluorescent nucleic acid-binding dyes are knownpigments used in textile and ink industries and are commerciallyavailable. References for preparations of these dyes can be found in theliterature. Many suitable reactive monomeric fluorescent non-nucleicacid dyes and non-fluorescent non-nucleic acid dyes are commerciallyavailable or may be prepared readily using known methods.

Synthesis of BRIDGE

BRIDGE is usually formed when the monomeric dyes are coupled to a bi- ortri-functional group, which is often commercially available. In general,the terminal portions of BRIDGE are from the monomeric dyes themselves,while the middle portion of BRIDGE is from a bi- or tri-functionalmolecule available from a commercial source. In some cases, asignificant portion of BRIDGE, such as up to about 90%, for example, maybe pre-attached to the monomeric dyes prior to the final assembly of thedimeric or trimeric dye. In some other cases, most of BRIDGE may beprepared separately before the monomeric dyes are attached. In the caseof heterodimer synthesis, a mono-protected bi-functional linker group isusually first attached to one monomeric dye, followed by de-protectionand coupling to the second monomeric dye. Hetero trimeric dyes may besynthesized using a similar stepwise protection-de-protection strategy.

Conjugation of the Monomeric Dyes to BRIDGE

In general, dimeric and trimeric dyes may be assembled by conjugatingmonomeric dyes having a suitable reactive group with a bi- ortri-functional linker in a one-step coupling reaction for some of thehomodimers, or in multi-step reactions for heterodimers and trimers orsome of the homodimers comprising multiple bridge element A. Examples ofsynthetic routes to selected homodimer and heterodimers areschematically illustrated in Scheme 6 directly below.

An example of the preparation of a homotrimeric dye is schematicallyillustrated in Scheme 7 directly below.

Examples of methods for preparing a dimeric dye having a reactive groupare illustrated in Scheme 8 directly below.

EXAMPLES Example 1 Preparation of 10-(3-Iodopropyl)acridine orange,iodide

One equivalent of 1,3-diiodopropane was added to a suspension of 5 g ofacridine orange (Aldrich) in 10 mL of chlorobenzene. The resultingmixture was stirred at 90-100° C. overnight. The hot reaction mixturewas poured into ˜200 mL of EtOAc. The orange precipitate was collectedby filtration and dried under vacuum, yielding ˜8 g.

Example 2 Preparation of 10-(5-Carboxypentyl)acridine orange, chloridesalt

10-(5-Ethoxycarbonylpentyl)acridine bromide was prepared using theprocedure of Example 1, with the exception that 1,3-diiodopropane wasreplaced with ethyl 6-bromohexanoic acid. The crude product (5 g) wassuspended in ˜100 mL methanol and 3 equivalents of NaOH dissolved in 30mL H₂O. The suspension was stirred at room temperature for 24 h.Methanol was removed by evaporation, and the remaining aqueous solutionwas acidified with concentrated HCl. About 50 mL saturated NaCl wasadded to precipitate the product. The product was collected byfiltration and then dried under vacuum at 45° C. for 24 hours.

Example 3 Preparation of DMAO (Dye No. 1 of Table 2)

10-(3-Iodopropyl)acridine orange, iodide (100 mg) was suspended in 20 mL2M dimethylamine in methanol in a sealed tube and then stirred at 60° C.overnight. The mixture was cooled to room temperature and then pouredinto 50 mL EtOAc. The precipitate was collected by centrifugation andthen dried under vacuum at 40° C. for 24 hours.

Example 4 Preparation of TMAO (Dye No. 2 of Table 2)

A mixture of DMAO (Dye No. 1 of Table 2) (11 mg, 0.023 mmol) and CH₃I(0.5 mL) in CH₃OH (2 mL) was refluxed gently for 4 days. The orangeproduct (10 mg) was collected by suction filtration.

Example 5 Preparation of PMAO (Dye No. 5 of Table 2)

A mixture of 10-(3-iodopropyl)acridine orange iodide salt (100 mg, 0.18mmol) and N,N,N′N′-tetramethyl-1,3-propanediamine (0.3 mL, 1.8 mmol) inCH₃OH (10 mL) was refluxed overnight. After cooling down to roomtemperature, the precipitate was collected by suction filtration. Theprecipitate was resuspended in CH₃OH (5 mL) and refluxed overnight andcollected by suction filtration. It was dried to a constant weight invacuo to give a dark red solid (14 mg).

Example 6 Preparation of AOAO-2Q (Dye No. 9 of Table 2)

A mixture of 10-(3-iodopropyl)acridine orange iodide salt (81 mg, 0.15mmol) and PMAO (100 mg, 0.15 mmol) in DMF (1.5 mL) was heated at 130° C.for 7 hours. After cooling down to room temperature, CH₃OH (15 mL) wasadded and the suspension was heated to reflux for 1 hour. Suctionfiltration gave the product as dark red solid (83.1 mg).

Example 7 Preparation of AOAO-2 (Dye No. 7 of Table 2)

Et₃N (0.15 mL, 1.05 mmol) and TSTU (320 mg, 1.05 mmol) were added to asuspension of 10-(5-carboxypentyl)acridine orange chloride salt (438 mg,1.03 mmol) in DMF (5 mL) at room temperature. The mixture was stirred atroom temperature for 15 minutes, followed by the addition of Et₃N (0.1mL) and 3,3′-diamino-N-methyldipropylamine (50 mg, 0.344 mmol). Afterthe mixture was stirred at room temperature overnight, EtOAc (20 mL) wasadded to precipitate the product. The crude product was re-dissolved inDMF and precipitated out again with EtOAc. The solid (250 mg) wasseparated by centrifugation.

Example 8 Preparation of AOAO-3 (Dye No. 8 of Table 2)

The dye (393 mg) was prepared by using the procedure to synthesizeAOAO-2 from 10-(5-carboxypentyl)acridine orange (432 mg, 1.03 mmol) andethylenediamine (25 mg, 0.42 mmol).

Example 9 Preparation of 10-(8-Bromooctyl)acridine orange bromide

A mixture of acridine orange (2 g, 7.53 mmol) and 1,8-dibromoactane (12mL, 67.8 mmol) in chlorobenzene (15 mL) was heated at 110° C. overnight.EtOAc (50 mL) was added and the suspension was refluxed for 1 hour.After cooling down to room temperature, suction filtration gave theproduct as orange solid (3.56 g).

Example 10 Preparation of AOAO-5 (Dye No. 11 of Table 2)

A mixture of 10-(8-bromoactyl)acridine orange bromide (0.5 g, 0.94 mmol)and acridine orange (0.3 g, 11.2 mmol) in DMF (5 mL) was heated at 130°C. overnight. EtOAc was added to precipitate the product. Repeatprecipitate from DMF and EtOAc gave the product as dark red solid (214mg).

Example 11 Preparation of AOAO-7 (Dye No. 13 of Table 2)

The dye (30 mg) was synthesized by using the procedure to make AOAO-2from 10-(5-carboxypentyl)acridine orange chloride salt (121 mg, 0.29mmol) and 1,5-diamino-pentane dihydrochloride (20 mg, 0.12 mmol).

Example 12 Preparation of AOAO-8 (Dye No. 15 of Table 2)

The dye (182 mg) was synthesized by using the procedure to make AOAO-2from 10-(5-carboxypentyl)acridine orange chloride salt (241 mg, 0.58mmol) and piperazine (20 mg, 0.23 mmol).

Example 13 Preparation of AOAO-11 (Dye No. 18 of Table 2)

The dye (112 mg) was synthesized by using the procedure to make AOAO-2from 10-(5-carboxypentyl)acridine orange chloride salt (180 mg, 0.43mmol) and 1,8-diamino-octane (25 mg, 0.17 mmol).

Example 14 Preparation of AOAO-12 (Dye No. 19 of Table 2)

The dye (76 mg) was synthesized by using the procedure to make AOAO-2from 10-(5-carboxypentyl)acridine orange chloride salt (147 mg, 0.35mmol) and 2,2′ oxybis(ethyl-amine) dihydrochloride (25 mg, 0.14 mmol).

Example 15 Preparation of AOAO-13 (Dye No. 20 of Table 2)

The dye (64 mg) was synthesized by using the procedure to make AOAO-2from 10-(5-carboxypentyl)acridine orange chloride salt (96 mg, 0.23mmol) and 4,7,10-trioxa-1,13-tridecanediamine (20 mg, 0.09 mmol).

Example 16 Preparation of1,3-Di-((2-(N-t-Boc-amino)ethyl)aminocarbonyl)benzene (Dye No. 101,shown directly below)

Et₃N (0.4 mL, 2.71 mmol) and TSTU (820 mg, 2.71 mmol) were added to asolution of isophthalic acid (220 mg, 1.32 mmol) in DMF (2 mL) at roomtemperature. The mixture was stirred at room temperature for 30 minutes.Addition of Et₃N (1 mL) and mono-tBoc-ethylenediamine (460 mg, 2.86mmol) followed. The mixture was stirred at room temperature overnightand then partitioned between 1N HCl (100 mL) and EtOAc (50 mL). Theaqueous layer was extracted with EtOAc (50 mL) and the combined organiclayers were washed with 1N HCl (2×50 mL), H₂O (50 mL), and saturatedNaCl (50 mL), and dried with anhydrous Na₂SO₄. The crude product waspurified by column chromatography using EtOAc:hexanes (9:1) as eluent togive the colorless solid product (356 mg).

Example 17 Preparation of 1,3-Di-((2-aminoethyl)aminocarbonyl)benzene,trifluoro-acetic acid salt (Dye No. 102, shown directly below)

1,3-di-((2-(N-t-Boc-amino)ethyl)aminocarbonyl)benzene (356 mg, 0.79mmol) was added to TFA (5 mL) at 0° C. The mixture was stirred at 0° C.for 1 hour and the solution was concentrated to dryness in vacuo. Thecolorless residue was dissolved in CH₃OH (2 mL) and added dropwise toEt₂O (30 mL). The precipitate was collected by centrifugation and driedto a constant weight in vacuo to give the solid product (425 mg).

Example 18 Preparation of AOAO-9 (Dye No. 16 of Table 2)

The dye (55 mg) was prepared by using the procedure to make AOAO-2 from10-(5-carboxypentyl)acridine orange chloride salt (109 mg, 0.26 mmol)and 1,3-Di((2-aminoethyl)aminocarbonyl)benzene, trifluoroacetic acidsalt (50 mg, 0.1 mmol).

Example 19 Preparation of1,3-Di((5-(N-t-Boc-amino)pentyl)aminocarbonyl)benzene (Dye No. 103,shown directly below)

The dye (555 mg) was prepared according to the procedure to make1,3-di-((2-(N-t-Boc-amino)ethyl)aminocarbonyl)benzene from isophthalicacid (254 mg, 1.53 mmol) and mono-tBoc cadaverine (640 mg, 3.15 mmol).

Example 20 Preparation of 1,3-Di-((5-aminopentyl)aminocarbonyl)benzene,trifluoro-acetic acid salt (Dye No. 104, shown directly below)

The dye (560 mg) was prepared according to the procedure for Dye No. 102(555 mg, 1.04 mmol).

Example 21 Preparation of AOAO-10 (Dye No. 17 of Table 2)

The dye (22 mg) was prepared by using the procedure to make AOAO-2 from10-(5-carboxypentyl)acridine orange chloride (95 mg, 0.23 mmol) and DyeNo. 104 (50 mg, 0.09 mmol).

Example 22 Preparation of AOAO-14 (Dye No. 21 of Table 2)

The dye (150 mg) was prepared by using the procedure to make AOAO-2 from10-(5-carboxypentyl)acridine orange chloride (166 mg, 0.40 mmol) anddiamido-dPEG-diamine (Quanta Biodesign of Powell, Ohio) (100 mg, 0.15mmol).

Example 23 Preparation of10-((((3-(N-Boc-amino)propyl)-N,N-dimethyl)ammonium)propyl) acridine,diiodide (Dye No. 105, shown directly below)

A mixture of 10-(3-iodopropyl)acridine orange iodide (500 mg, 0.89 mmol)and 3-(N-t-Boc-amino)propyl-N,N-dimethylamine (1.8 g, 8.9 mmol) in CH₃OH(50 mL) was refluxed overnight. After cooling down to room temperature,the precipitate was collected by suction filtration and dried to aconstant weight to give Dye No. 105 as an orange solid (292 mg).

Example 24 Preparation of10-((((3-ammonium)propyl)-N,N-dimethyl)ammonium)propyl acridine,trifluoroacetate salt (Dye No. 106, shown directly below)

Dye No. 105 (50 mg, 0.06 mmol) was added to TFA (2 mL) at 0° C. Themixture was stirred at 0° C. for 30 minutes. The solution wasconcentrated to dryness in vacuo and the residue was dissolved in CH₃OH(3 mL). The solution was added dropwise to Et₂O (30 mL) and theprecipitate was collected by centrifugation and dried to a constantweight in vacuo to give Dye No. 106 as an orange solid (28 mg).

Example 25 Preparation of AOAO-4 (Dye No. 10 of Table 2)

The dye (23 mg) was prepared by using the procedure to make AOAO-2 from10-(5-carboxypentyl)acridine orange chloride salt (31 mg, 0.075 mmol)and Dye No. 106 (28 mg, 0.036 mmol).

Example 26 Preparation of 10-(6-(N-Phthalimido)hexyl)acridine orangebromide salt (Dye No. 107, shown directly below)

A mixture of acridine orange (2 g, 7.54 mmol) andN-(6-bromohexyl)phthalimide (4.7 g, 15.1 mmol) in chlorobenzene (20 mL)was heated at 110° C. for 2 days. EtOAc (50 mL) was added and thesuspension was heated to reflux for 1 hour. After cooling down to roomtemperature, the product Dye No. 107 was collected by suction filtrationas an orange solid (3.76 g).

Example 27 Preparation of10-(5-((5-Carboxypentyl)aminocarbonyl)pentyl)acri-dine orange, iodide(Dye No. 108, shown directly below)

Et₃N (40 μL, 0.28 mmol) and TSTU (81 mg, 0.27 mmol) were added to asuspension of 10-(5-carboxypentyl)acridine orange chloride (107 mg,0.258 mmol) in DMF (3 mL). The mixture was stirred at room temperaturefor 15 minutes. Addition of Et₃N (0.2 mL) and a solution of6-aminohexanoic acid (67 mg, 0.51 mmol) in H₂O (1 mL) followed. Themixture was stirred at room temperature for 1 hour and concentrated todryness in vacuo. The residue was triturated with H₂O to give Dye No.108 as an orange solid (125 mg).

Example 28 Preparation of 9-Cyano-10-(5-carboxypentyl)acridine orange,chloride (Dye No. 109, shown directly below)

A mixture of 10-(5-carboxypentyl)acridine orange (0.15 g, 0.361 mmol)and sodium cyanide (35 mg, 0.722 mmol) in DMF (3 mL) was stirred at roomtemperature in open air for 2 days. CH₃CN (10 mL) was added and theresulting suspension was stirred at room temperature for 1 hour. Thedark blue precipitate was collected by centrifugation and dried to aconstant weight in vacuo to give Dye No. 109 (130 mg).

Example 29 Preparation of AOAO-12R (Dye No. 22 of Table 2)

Et₃N (32 μL, 0.23 mmol) and TSTU (68 mg, 0.227 mmol) were added to asolution of Dye No. 109 (98.3 mg, 0.223 mmol) in DMF (2 mL) at roomtemperature. The mixture was stirred at room temperature for 15 minutes.Addition of Et₃N (100 μL) and 2,2′-oxybis-(ethylamine) dihydrochloride(16 mg, 0.09 mmol) followed. The mixture was stirred at room temperaturefor 2 days. The solution was concentrated to about 1 mL and EtOAc (2 mL)was added. The precipitate was collected by centrifugation. The productwas re-dissolved in DMF and precipitated again with EtOAc to give DyeNo. 22 as a dark blue solid (54.4 mg).

Example 30 Preparation of 9-Aminocarbonyl-10-(5-carboxyphentyl)acridine(Dye No. 110, shown directly below)

A solution of Dye No. 109 (30 mg, 0.068 mmol) in 75% H₂SO₄ (1 mL) washeated at 60° C. for 2 days. After cooling down to room temperature, themixture was added to Et₂O (10 mL). The precipitate was collected bycentrifugation and re-dissolved in CH₃OH (1.5 mL). EtOAc (10 mL) wasadded and the solid precipitate was collected by centrifugation anddried to a constant weight in vacuo to give Dye No. 110 as a dark pinksolid (20.4 mg).

Example 31 Preparation of N-Carboxypentyl Thiazole Orange (ShownDirectly Below)

The dye was prepared using published procedure (Carreon, et al., Org.Let. 6(4), 517 (2004)).

Example 32 Preparation of TOTO-3 (Dye No. 14 of Table 2)

The dye (354 mg) was prepared using the procedure to synthesize AOAO-2from N-Carboxypentyl thiazole orange (460 mg, 1.04 mmol) and ethylenediamine (25 mg, 0.42 mmol).

Example 33 Preparation of10-(5-((2-(N-t-Boc-amino)ethyl)aminocarbonyl)pentyl)acridine orangechloride salt (Dye No. 111, shown directly below)

Et₃N (106 μL, 0.76 mmol) and TSTU (230 mg, 0.76 mmol) were added to asuspension of 10-(5-carboxypentyl)acridine orange chloride (302 mg, 0.73mmol) in DMF (3 mL). The mixture was stirred at room temperature for 15minutes. The addition of Et₃N (350 μL) and mono t-BOC-ethylenediamine(150 mg, 0.92 mmol) followed. The mixture was stirred at roomtemperature for 1 hour and then concentrated to dryness in vacuo. Theresidue was dissolved in CH₃CN (2 mL) and precipitated by the additionof EtOAc (20 mL). The precipitate was collected by centrifugation anddried to a constant weight to give Dye No. 111 as orange solid (365 mg).

Example 34 Preparation of10-(5-((2-Ammoniumethyl)aminocarbonyl)pentyl)acridine orange,trifluoroacetate (Dye No. 112, shown directly below)

Dye No. 111 (347 mg, 622 mmol) was added in one portion totrifluoroacetic acid (3 mL) at 5° C. The mixture was stirred at 5° C.for 1 hour and concentrated to dryness in vacuo. The residue wasdissolved in CH₃OH (3 mL) and added dropwise to Et₂O (50 mL). Theprecipitate was collected by centrifugation to give Dye No. 112 asorange solid (297 mg).

Example 35 Preparation of AOTO-3 (Dye No. 23 of Table 2)

Et₃N (20 μL, 0.142 mmol) and TSTU (42.2 mg, 0.142 mmol) were added to asolution of N-carboxypentylthiazole orange (62 mg, 0.142 mmol) in DMF (1mL). The mixture was stirred at room temperature for 15 minutes.Addition of Et₃N (70 mL) and Dye No. 112 (50 mg, 0.095 mmol) followed.The mixture was stirred at room temperature for 2 hours and thenconcentrated to dryness in vacuo. The residue was re-dissolved in DMF (1mL) and EtOAc (2 mL) was added. The precipitate was collected bycentrifugation. Repeated precipitation from DMF and EtOAc gave theproduct as orange red solid (50.4 mg)

Example 36 Preparation of TOTO-12 (Dye No. 24 of Table 2)

The dye (19.4 mg) was prepared by using the procedure to synthesizeAOAO-2 from N-carboxypentylthiazole orange (94.5 mg, 0.2145 mmol) and2,2′ oxybis(ethylamine) dihydrochloride (15 mg, 0.085 mmol).

Example 37 Preparation of TO (3)TO (3)-12 (Dye No. 25 of Table 2)

The dye (32.6 mg) was prepared by using the procedure to synthesizeAOAO-2 from N-carboxypentyl thazole blue (Carreon, et al., Org. Let.6(4), 517 (2004); and Benson, et al., Nucleic Acid Res. 21(24), 5727(1993)) (99 mg, 0.212 mmol) and 2,2′ oxybis(ethylamine) dihydrochloride(15 mg, 0.085 mmol).

Example 38 Preparation of TO(3)TO(3)-2 (Dye No. 26 of Table 2)

The dye (28.4 mg) was prepared by using the procedure to synthesizeAOAO-2 from N-carboxypentyl thiazole blue (76 mg, 0.173 mmol) and3,3′-diamino-N-methyldi-propylamine (10 mg, 0.069 mmol).

Example 39 Preparation of10-(5-((5-(N-t-Boc-amino)pentyl)aminocarbonyl)pentyl)-acridine orange,chloride (Dye No. 113, shown directly below)

The dye (280 mg) was prepared by using the procedure to synthesize DyeNo. 111 from 10-(5-carboxypentyl)acridine orange chloride (200 mg, 0.483mmol) and mono t-BOC-cadaverine (130 mg, 0.628 mmol).

Example 40 Preparation of10-(5-((5-ammoniumpentyl)aminocarbonyl)pentyl)acridine orange,trifluoroacetate (Dye No. 114, shown directly below)

Dye No. 114 (234 mg) was prepared by using the procedure to synthesizeDye No. 112 from Dye No. 113 (280 mg, 0.467 mmol).

Example 41 Preparation of AORO-7 (Dye No. 27 of Table 2)

The compound (29 mg) was prepared by using the procedure to synthesizeAOTO-3 from compound No. 114 (35 mg, 0.061 mmol) and the rosamine dye(Biotium, Inc. (Hayward, Calif.)) (40 mg, 0.063 mmol).

Example 42 Absorbance and fluorescence of DMAO and AOAO-7

The absorbance spectra, as shown in FIG. 2 and FIG. 3, and fluorescenceemission spectra, as shown in FIG. 4, of DMAO and AOAO-7, were measuredseparately without DNA presence, or with DNA presence (2 mg/ml of salmonsperm DNA), in PBS buffer. All dye concentrations were adjusted toprovide an optical density of 0.05 at 495 nm. The spectra werenormalized to 1 in the absorbance plot. Relative to DMAO, AOAO-7exhibits a new shorter wavelength peak at 471 nm in absorbance,indicating aggregation of the two acridine monomers within AOAO-7. Uponbinding to DNA, absorbances of AOAO-7 and DMAO showed 5 nm- and 10nm-red shifts, respectively, relative to free dyes. The fluorescence offree AOAO-7 is about 5 times lower than that of DMAO. The lowerbackground fluorescence of AOAO-7 is advantageous in real time qPCR. Thefluorescence per acridine monomer of AOAO-7 is close to that of DMAO,indicating that two monomers of AOAO-7 no longer quenched each otherwhen bound to DNA and the linker between the two did not exhibitnegative effect on the quantum yield.

Example 43 Absorbance spectra of TOTO-1 and TOTO3

In a similar manner to that described in connection with Example 42, theabsorbance spectra of TOTO-1 and TOTO-3 were measured without DNApresence, as shown in FIG. 5, or in the presence of 2 mg/ml of salmonsperm DNA, as shown in FIG. 6, in PBS buffer. As shown in FIG. 5, thespectra of the free dyes indicate that TOTO-3, which has BRIDGE that isneutral or substantially devoid of positive charges, forms anintramolecular H-dimer, or a hairpin structure, while TOTO-1, which hasmultiple positive charges, has less spectral shift. As shown in FIG. 6,the absorption spectra of both TOTO-1 and TOTO-3 in the presence of DNAshift to about the same position, indicating that the hairpin structureof TOTO-3 dimer opens up upon binding to DNA, and that both TOTO-1 andTOTO-3 form similar types of DNA-dye complexes.

Example 44 Fluorescence of AOAO-12 in response to different amount ofDNA

The fluorescence of 0.1 μM AOAO-12 in 200 mL of PBS in the presence of0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 2.0, 4.0, 6.0, 8.0 and 10 μg/ml finalconcentrations of salmon sperm DNA or a mixture of single-stranded 20meroligonucleotide were measured on a microtiter plate reader (SpectraMaxof Molecular Devices Corporation (Sunnyvale, Calif.)). The fluorescencewas plotted against DNA concentration, as shown in FIG. 7. It can beseen that fluorescence linearly responded to DNA up to 2.0 μg/ml(inset). At higher concentrations of DNA, the response becamenon-linear. AOAO-12 fluoresces more intensely when bound to doublestranded DNA than when bound to single stranded DNA.

Example 45 Comparing signal strengths of AOAO-12 and SYBR Green I inqPCR

This example demonstrates the superior property of AOAO-12 to SYBR GreenI in fluorescence signal strength in qPCR. All real-time amplificationswere performed in 20 μL reaction solution comprising 10 mM Tris (pH8.0), 50 mM KCl, 3.5 mM MgCl2, 2 mM each of dNTP, and 1 unit of AmpliTaqDNA polymerase (ABI, Foster City, Calif.) and various concentrations ofa fluorescent monitoring dye. An ATPB fragment (SEQ ID NO: 1) in pTOPOplasmid was amplified with 0.5 μM forward primer5′-GAGGTCTTCACAGGTCATA-3′ (SEQ ID NO: 2), 0.5 μM reverse primer5′-CTCTTCAGCCAGCTTATC-3′ (SEQ ID NO: 3). The thermal regimen was set at95° C. for 1 minute followed by 50 cycles of 15-second duration at 95°C., of 15-second duration at 55° C., and of 15-second duration at 72° C.Fluorescence was measured at the 55° C. stage. Consistent with anearlier report (Nath, K., et al., Effects of ethidium bromide and SYBRGreen I on different polymerase chain reaction systems, J. BiochemBiophys Methods 42, 15 (2000)), SYBR Green I exhibited inhibition to PCRreactions, as shown in FIG. 8, where Ct was delayed at higher SYBR GreenI concentrations. Relative to SYBR Green I concentrations, higherconcentrations of AOAO-12 could be added to reactions without exhibitinginhibition. As a result, the final fluorescence strength with AOAO-12could be several folds higher, allowing for more sensitive detection.Alternatively, with AOAO-12, a less sensitive optical device could beused in thermal cycling fluorometers, leading to less expensiveinstruments.

Example 46 Titration of Human Genomic DNA

Amplifications of a UBC fragment (SEQ ID NO: 8) from human genomic DNAwere performed under conditions similar to those set forth in Example45, with either SYBR Green I (final absorption peak at 495 nmcorresponds to an optical density of 0.025, or A₄₉₅=0.025) or AOAO-12(final A₄₉₅=0.1), except that (1) different forward and reverse primerssets (5′-ACTGGTAAGACCATCACC-3′ (SEQ ID NO: 9) and5′-GCAATGAAATTTGTTGAA-3′ (SEQ ID NO: 10)) were used, and (2) a series of10-fold dilutions of human DNA served as the templates. FIG. 8 showsamplification plots of the reactions starting with 10⁵ copies of humanDNA down to 10 copies either with SYBR Green I or with AOAO-12. Theinset shows that the Ct value is reversibly correlated with thelogarithm of starting copy number monitored with both dyes. AOAO-12 isevidently superior to SYBR Green I in signal strength.

Example 47 TO, TOTO-1 and TOTO12 in qPCR

This example demonstrates the improved property of TOTO-12 over TO(thiazole orange, an asymmetric cyanine dye), or TOTO-1 in qPCR. Allreal-time amplifications were performed as in Example 5, except that TO,TOTO-1 and TOTO-12 were used. As shown in FIG. 11, TOTO-1 completelyinhibited the PCR reaction, while TOTO-12 gave improved Ct and improvedfluorescence intensity relative to TO in qPCR. The TOTO-12 dimeric dyeis superior to each of the TO and TOTO-1 dyes.

Example 48 Absorbance and fluorescence of AORO-7

In a manner similar to that described in connection with Example 42, theabsorbance spectra of AORO-7 were taken alone or in the presence of 2μg/ml of salmon sperm DNA in PBS buffer, as shown in FIG. 12. Theemission spectra of AORO-7 bound to DNA are shown in FIG. 13. Theheterodimeric dye AORO-7 comprises a monomeric AO dye and a fluorescentnon-nucleic acid rosamine RO dye. Two emission peaks, contributed by AOand RO moieties, respectively, were recorded. It was possible to recordqPCR with AORO-7 in Channel 1 with the excitation set at 490 nm and theemission collected at 530 nm, or Ex490/Em530, or in Channel 3 withEx580/Em630, on a Chromo4 from Bio-Rad Laboratories (Hercules, Calif.),but only weakly in Channel 2 (Ex520/Em570) (data not shown). As thefluorescence resonance energy transfer (FRET) effect is negligible,either monomeric dye constituent, AO or RO, can be chosen as thereporter dye.

Example 49 Heterodimer dye AORO-7 in qPCR

The example demonstrates the utility of AORO-7 in qPCR. Real timeamplifications were performed as in Example 5, except that AORO-7 withan optical density of 0.025 at 600 nm in the final solution was used. Asshown in FIG. 14, AORO-7 may be used effectively be used to monitor theqPCR reaction course and to detect the melting curve of the amplicon(inset). The finding that this qPCR was monitored in Channel 3(Ex580/Em630) on an iCycle IQ Multiple-Color Real-Time PCR DetectionSystem from Bio-Rad Laboratories (Hercules, Calif.) provides an examplethat almost any dye of desirable wavelength may be tailored into a DNAreporter dye in qPCR by methods disclosed herein, thus widening thespectral use for qPCR monitoring. At present, most TaqMan probes use FAMor VIC, which occupy the same channels of SYBR Green I. It would beadvantageous to use a sequence-specific-probe, such as a TaqMan probe,and a sequence-non-specific dye of different wavelength, such as AORO-7,in qPCR, wherein the probe provides sequence specificity and the dyeprovides other parameters of the amplicon, such as melting temperautres.

Example 50 Melting peaks monitored with AOAO-12

SYBR Green I was reported to be advantageous in that the meltingtemperature of an amplicon could be detected after qPCR amplification.The melting temperature provides valuable information about theamplicon, as it is a function of the size and GC content of theamplicon. No melting temperatures could be collected from TaqManreactions. Melting curves from four amplicons, i.e., HMBS (SEQ ID NO:4), RPL4 (SEQ ID NO: 5), SDHA (SEQ ID NO: 6), and TBP (SEQ ID NO: 7)were measured from reactions amplified in the presence of AOAO-12 (PanelA) or SYBR Green (Panel B) and are presented in FIG. 15. The meltingpeaks collected with AOAO-12 correlated well with those collected withSYBR Green I. As AOAO-12 binds DNA with high affinity but less tightlythan SYBR Green I, melting temperatures from AOAO-12 are about 2 degreeslower than those from SYBR Green I. As higher concentrations of AOAO-12could be used in real time PCR reactions, melting peaks are markedlyhigher. The data demonstrate the relative utility of AOAO-12.

It has been reported that qPCR reactions with SYBR Green I tend to formprimer-dimers, and the tendency is closely related to the concentrationof SYBR Green I. It has been postulated that SYBR Green I binds to DNAso tightly that it interferes with the performance of Taq DNApolymerase. As AOAO-12 has less affinity to DNA, the interference shouldbe alleviated. This property of AOAO-12 is evident from FIG. 15, in thatthe HMBS amplification reaction in the presence of SYBR Green I has anextra primer dimer peak, while the same amplification in the presence ofAOAO-12 exhibits only a single, clean, and specific peak.

Example 51 Stability of AOAO dyes

The stability of dimeric dyes comprising monomeric AO was demonstrated.Specifically, AOAO-12 was kept in PCR reaction buffers with PCRproducts. The mixture was heated to 96° C. for 40 minutes. During theheating course, the mixture was brought down to 60° C. briefly tomonitor the fluorescence. As shown in FIG. 16, AOAO-12 is stable at 96°C. for 40 minutes substantially without loss of fluorescence. The datademonstrate the robustness of AOAO-12 in PCR.

Example 52 Preparation of TOTO-13 (Dye No. 29 of Table 2)

The dye (102 mg) was prepared using the procedure to synthesize AOAO-2(Dye No. 7 of Table 2) from N-carboxypentyl thiazole orange (102 mg,0.23 mmole) (Example 31) and 4,7,10-trioxa-1,13-tridecanediamine (23 mg,0.1 mole).

Example 53 Preparation ofN-(5-carboxypentyl)-4-(4-(dimethylamino)styryl)pyridinium bromide

A mixture of 4-N,N-dimethylaminobenzaldehyde (3 g, 20 mmoles),N-(5-carboxy-pentyl)picolinium bromide (5.6 g, 20 mmoles) and piperidine(2 mL) in ethanol (100 mL) was heated at 60° C. overnight. The mixturewas evaporated to dryness in vacuo. The residue was redissolved inmethanol and then precipitated with ether to give the product (6.7 g).

Example 54 Preparation of STST-19 (Dye No. 31 of Table 2)

The dye (85 mg) was prepared using the procedure to prepare AOAO-2(Example 7) fromN-(5-carboxypentyl)-4-(4-(dimethylamino)styryl)pyridinium bromide(Example 53) (200 mg, 0.5 mmole) and 2,2′-oxybis(ethylamine)dihydrochloride (36 mg, 0.2 mmoles).

Example 55 Preparation of STST-27 (Dye No. 30 of Table 2)

The dye (81.8 mg) was prepared using the procedure to prepare AOAO-2(Example 7) fromN-(5-carboxypentyl)-4-(4-(dimethylamino)styryl)pyridinium bromide(Example 53) (200 mg, 0.5 mmole) and 4,7,10-trioxa-1,13-tridecanediamine(44 mg, 0.2 mmoles).

A useful dye, such as a dimeric dye or a trimeric dye, has beendescribed herein. Such a dye may have any of a number of desirableproperties, such as relatively low background fluorescence, relativelylow PCR inhibition, good fluorescent signal strength, and goodstability, for example. Generally, a dye having at most one positivecharge may have any of a number of applications, such as use in thelabeling of another molecule, and such as use in the detection of thepresence or absence of nucleic acid, for example. Further, generally,such a dye that is substantially neutral, has any of a number ofapplications, such as use in PCR processes or use in the detection ofthe presence or absence of nucleic acid, or use in quantitativereal-time PCR, for example.

A number of useful dimeric and trimeric dyes have been described. By wayof example, a dye that is suitable for covalent conjugation with, orlabeling of, another molecule to confer the nucleic acid-detectingcapability of the dye on the molecule; a dye that is suitable fordetecting the presence or absence of nucleic acid in a sample that mayor may not comprise nucleic acid; and a dye that is suitable fordetecting nucleic acid formation or a lack thereof in a sample, such asa sample that undergoes a process suitable for nucleic acidamplification should the sample comprise a target nucleic acid, havebeen described. A method for preparing any of the foregoing dyes and amethod of using any of the foregoing dyes have also been described. Anymethod of using a composition described herein is contemplated herein. Akit suitable for determining nucleic acid formation or a lack thereof ina sample, which comprises a suitable dye of the present invention, and asuitable composition sufficient for amplification of a target nucleicacid in a sample should it comprise a target nucleic acid, iscontemplated herein, as is any kit comprising a composition describedherein that has useful application.

Various modifications, processes, as well as numerous structuresrelating to the description herein may be applicable, as will be readilyapparent to those of ordinary skill in the art, upon review of thespecification. Various references, publications, provisional andnon-provisional United States or foreign patent applications, and/orUnited States or foreign patents, have been identified herein, each ofwhich is incorporated herein in its entirety by this reference. Variousaspects and features may have been explained or described in relation tounderstandings, beliefs, theories, underlying assumptions, and/orworking or prophetic examples, although it will be understood that anysuch understanding, belief, theory, underlying assumption, and/orworking or prophetic example is not binding. Although the variousaspects and features have been described with respect to variousembodiments and examples herein, it will be understood that any of sameis not limiting with respect to the full scope of the appended claims.

The invention claimed is:
 1. A method of performing a nucleic acidamplification reaction comprising: (i) providing a reaction mixturecomprising said sample and reagents necessary for amplifying nucleicacid; (ii) subjecting said reaction mixture to a polymerization reactionunder conditions suitable for formation of amplified nucleic acid; (iii)adding a fluorescent nucleic acid dye, wherein said addition takes placebefore, during, or after step (ii), and wherein said dye has theformula:

wherein at least one of Q₁ and Q₂ is a fluorescent nucleic acid dyeconstituent; and R_(r) is a reactive group for covalent attachment to anucleic acid; (iv) illuminating the reaction mixture with light; and (v)detecting fluorescence emission from said reaction mixture, wherein saidemission is indicative of the presence of nucleic acid, wherein B is anucleotide or an oligonucleotide; BRIDGE is a substantially aliphaticlinker comprising from about 15 to about 150 non-hydrogen atoms and upto one positive charge; and each of Q₁ and Q₂ is independently anacridine dye, an asymmetric cyanine dye, a symmetric cyanine dye, aphenanthridinium dye, a pyronin dye, and a styryl dye, and whereinBRIDGE comprises:-L₁-[A¹-(CH₂)_(α)-]_(a)[A²-(CH₂)_(β)-]_(b)[A³-(CH₂)_(γ)-]_(c)[A⁴-(CH₂)_(δ)-]_(d)[A⁵-(CH₂)_(ε)-]_(e)[A⁶-(CH₂)_(ζ)-]_(f)[A⁷-(CH₂)_(η)-]_(g)[A⁸-(CH₂)_(θ)-]_(h)[A⁹-(CH₂)_(τ)-]_(i)-A¹⁰-L₂- wherein each of L₁ andL₂, independently, is a moiety comprising a single bond; a polymethyleneunit having 1 carbon to about 12 carbons, inclusive, optionallycomprising at least one hetero atom selected from N, O and S; or an aryloptionally comprising at least one hetero atom selected from N, O and S;each of A¹, A², A³, A⁴, A⁵, A⁶, A⁷, A⁸, A⁹, and A¹⁰, independently, is anucleic-acid-binding-enhancing-group (NABEG); a branched alkyloptionally comprising at least one hetero atom selected from N, O and S;or at least one saturated 5- or 6-membered ring, optionally comprisingat least one hetero atom selected from N, O and S; each of α, β, γ, δ,ε, ζ, η, θ, and τ, independently, is zero or an integer from 1 to about20, inclusive; each of a, b, c, d, e, f, g, h, and i, independently, iszero or an integer from 1 to about 20, inclusive;

and one of A¹, A², A³, A⁴, A⁵ A⁶, A⁷, A⁸, A⁹, and A¹⁰ has the formula:

wherein T is a substituted carbon, a substituted nitrogen, or an aryloptionally comprising at least one hetero atom selected from O, N and S;L₃ is covalently bound to R_(r) or B and is a moiety comprising a singlebond; a polymethylene unit having 1 carbon to about 12 carbons,inclusive, optionally comprising at least one hetero atom selected fromN, O and S; or an aryl optionally comprising at least one hetero atomselected from N, O and S; and B′ has the formula:—(CH₂)_(α′)-[A¹¹-(CH₂)_(β′)-]_(a′)[A¹²-(CH₂)_(γ′)-]_(b′)[A¹³-(CH₂)_(δ′)-]_(c′)A¹⁴-,wherein —(CH₂)_(α′) is covalently linked to T and A¹⁴ is covalentlylinked to L₃; each of A¹¹, A^(l2), A¹³, and A¹⁴ is independently anucleic-acid-binding-enhancing-group (NABEG), a branched alkyloptionally comprising at least one hetero atom selected from N, O and S;or at least one saturated 5- or 6-membered ring optionally comprising atleast one hetero atom selected from N, O and S; each of α′, β′, γ′, andδ′ is independently zero or an integer from 1 to about 20, inclusive;and each of a′, b′, and c′ is independently zero or an integer from 1 toabout 20, inclusive.
 2. The method of claim 1, wherein the fluorescentnucleic acid dye has the structure:


3. The method of claim 1, wherein T is a substituted nitrogen.
 4. Themethod of claim 1, wherein each of Q₁ and Q₂ is independentlyrepresented by the structure:

wherein each of R₁ or R₁′ of is H; alkyl or alkenyl having 1 carbon to 6carbons, inclusive; a halogen; —OR₉; —SR₁₀; —NR₁₁R₁₂; —CN; —NH(C═O)R₁₃;—NHS(═O)₂R₁₄; —C(═O)NHR₁₅; or a substituent associated with minor groovebinding; or represents where BRIDGE attaches to the structure; when R₁or R₁′ of comprises at least one of R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅,any said one of R₉, R₁₀, R₁₁, R₁₂, R₁₃, R₁₄ and R₁₅, independently, is Hor alkyl having 1 carbon to 12 carbons, inclusive, optionallyincorporating 1 to 2 nitrogen(s), inclusive, or an aryl; when R₁ or R₁′of comprises R₁₁ and R₁₂, R₁₁ and R₁₂ may in combination form a 5- or6-membered, saturated or unsaturated ring, which optionally comprises atleast one hetero atom selected from N and O; R₂ is methyl or ethyl, orrepresents where BRIDGE attaches to the structure; X is selected from Oand S; n is selected from 0, 1, and 2; R₆ is H; alkyl or alkenyl having1 carbon to 10 carbons, inclusive, optionally comprising at least onehetero atom selected from N, O, and S; a halogen; —OR₁₆; —SR₁₆;—NR₁₆R₁₇; or a substituted or an unsubstituted aryl, optionallycomprising 1 to 3 hetero atom(s), inclusive, selected from N, O, and S;or represents where BRIDGE attaches to the structure; R₇ is H; alkyl oralkenyl having 1 carbon to 10 carbons, inclusive, optionally comprisingan aryl and at least one hetero atom selected from N, O, and S; or asubstituted or an unsubstituted aryl optionally comprising 1 to 3 heteroatom(s), inclusive, selected from N, O, and S; or represents whereBRIDGE attaches to the structure; R₈ and R₈′ in combination form a fusedaromatic ring, which may be further substituted 1 to 4 time(s),inclusive, independently, by C1-C2, inclusive, alkyl, C1-C2, inclusive,alkoxy, C1-C2, inclusive, alkylmercapto, or a halogen; each of R₁₆ andR₁₇ independently is H; alkyl having 1 carbon to 12 carbons, inclusive,optionally incorporating 1 to 2 nitrogen(s) or an aryl; or R₁₆ and R₁₇may in combination form a 5- or 6-membered saturated or unsaturatedring, which optionally comprises at least one hetero atom selected fromN and O; only one of R₁′, R₂, R₆, and R₇ represents where BRIDGEattaches to the structure; and ψis an anion.
 5. The method of claim 1,wherein each of Q1 and Q2 is independently represented by the structure:

wherein R₇ represents where BRIDGE attaches to the structure; and ψ isan anion.
 6. The method of claim 1, wherein each of Q1 and Q2 isindependently represented by the structure:

wherein R₉ represents where BRIDGE attaches to the structure; and ψ isan anion.
 7. The method of claim 1, wherein each of Q1 and Q2 isindependently represented by the structure:

wherein each R₁ is independently H or a C1-C2, inclusive, alkyl; one ofR₂ and R₃ represents where BRIDGE attaches to the structure; when R₂represents where BRIDGE attaches to the structure, R₃ is H or —CH₃; whenR₃ represents where BRIDGE attaches to the structure, R₂ is selectedfrom H, —CH₃, —NH₂, —NHCH₃, —CN, and —C(═O)NH₂; each R₆ is independentlyH or a C1-C2, inclusive, alkyl; each R₇ is independently H or a C1-C2,inclusive, alkyl; for each pair of adjacent R₆ or R₇ and R₁,independently, R₆ or R₇ and R₁ may in combination form a 5- or6-membered, saturated or unsaturated ring; and ψ is an anion.